Periodic Reporting for period 1 - CYgnaling (Probing the role of orphan Cytochrome P450 oxygenases in signaling compounds biosynthesis in plants by a comparative genomics and gene conservation approach)
Reporting period: 2016-05-01 to 2018-04-30
Plant growth regulators, such as phytohormones, are key factors for communication between parts of the plant, allowing a coordinated response of the whole plant to environmental challenges. These phytohormones are produced by specific biosynthetic pathways involving a cascade of enzymatic reactions. Currently, only a limited number of compounds have been characterized as plant growth regulators. They usually have drastic effects on plant development and have, for most of them, been known for many years. However, beside these players, recent works show that other signaling compounds with more subtle effects are also necessary for normal plant growth. The chemical natures of these compounds are yet unknown. In this project, we propose to study biosynthesis and action of overlooked plant hormones and signaling pathways using a reverse genetic approach in the model plant Arabidopsis thaliana (mouse-ear cress). In particular, we are focusing on genes coding for oxygenase enzymes belonging to the cytochrome P450 (P450) family. The P450 family plays a pivotal role in the known phytohormone biosynthesis or degradation, as all of their biosynthetic pathways, except one, require at least one P450 action to form a bioactive compound.
Our main objectives in this project is (1) to select candidate genes and test their implication on plant development as a putative actor in phytohormone biosynthesis, (2) identify the chemical compounds that are being produced by the P450s of interest and more generally (3) to apprehend how the putative signaling pathways regulates plant growth.
To assess the enzymatic activities of our candidate cytochrome P450s, and thus better understand the chemical nature of the putative dependent signaling compounds, we produced the recombinant proteins in recombinant systems and assess their activities in vitro. A main part of the work was dedicated to optimize the protein productions as they prove difficult to produce. When proteins could be produced, we did not detect an in vitro activity with the substrates tested so far. In parallel, we have done metabolic analyses of our mutants to detect any compounds that might differentially accumulate in plant tissues compared to control plants. With these experiments we found several candidate compounds which chemical nature need to be experimentally confirmed.
During the course of this project, we produced the following results:
1. Streamlined method for bioinformatic analysis of P450 gene evolution
2. Selection of several gene of interest for functional analysis based on specific evolutionary signature.
3. Produced a fine map of gene-of-interest expression domains
4. Produced and discovered of a novel mutant exhibiting a hormone-like phenotype
5. Found several compounds differentially accumulated in our mutant of interest
6. Generated differential mutant versus wild-type transcriptome data
7. Produced recombinant proteins for in vitro activity.