• Arabidopsis
• PCO
• submergence
• synthetic biology
• yeast

In normoxic conditions, PCO (Plant Cysteine Oxidase) enzymes catalyse the oxidation of ERF-VII (Ethylene Response Factors) transcription factors. This is an oxygen-dependent step of the N-degron pathway that will lead to the degradation of the ERF-VIIs. In hypoxia, low levels of oxygen lower PCO activity, ERF-VIIs are stabilized and can initiate the expression of genes involved in the hypoxic response.
The engineering of PCOs has gained increasing interest due to the greater extremes of stress, such as submergence, that plants have to face as a result of climate change. Our aim is identify PCO variants with reduced activity, leading to faster stabilization of ERF-VIIs resulting in a better response to submergence.
To address this, different strategies were used. We first followed a rational design, and tested different PCO4 variants that could hinder its activity due to structural variations either in the oxygen tunnel or its active site. We also set-up preliminary experiments to exploit directed evolution based on random mutagenesis, using yeast as a host.
We tested two pre-existing variants, already shown biochemically to reduce PCO activity, in Arabidopsis thaliana by submerging them for 2.5 and 3.5 days and comparing their survival and recovery rate with those of the wild-types. The two variants showed higher survival rates, consistent with previous experiments and biochemical evaluations.
For functional assessment, we implemented a method for the high-throughput screening of PCO variants activity based on luminescence in yeast. The activity of each variant was measured by comparing the relative Fluc activity after 6 hours of (aerobic or hypoxic) treatment with that of the wild type and a negative control. 12 PCO variants of interest were tested with this in vivo platform.
This yeast assay is a useful tool for the rapid, cost-effective, large-scale screening of PCO variants in an in vivo platform and in a controlled environment before moving to more complex plant models.
Finally, we developed a construct for the selection in yeast of interesting PCO variants using a previously characterized system based on zeocin resistance and 5FdU susceptibility. This system can be used to find novel interesting PCO variants with a high-throughput selection from a pool of randomly mutagenized PCOs, solely based on their activity. This approach can also facilitate the discovery of new residues of interest for manipulating PCO activity.
These approaches provide valuable tools to further investigate the functionality of PCOs through both random and rational methods