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Probing the role of orphan Cytochrome P450 oxygenases in signaling compounds biosynthesis in plants by a comparative genomics and gene conservation approach

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 are sessile organisms and thus permanently need to modulate their growth in order to optimize their exposure to light or access to nutrients. They also need to efficiently react to the pathogenic pressure. In agriculture in particular, plant growth, adaptation and resistance is a major challenge as they directly impact the yield and the quality of the output food. Better understanding the fine regulation leading to plant fitness is thus an important undertaking with huge implication in real-life economy.
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.
During this project we have done an extensive bioinformatic work to select several candidate genes from the plant Arabidopsis thaliana showing a phylogenomic signature similar to the one observed for known genes involved in hormones biosynthesis. A fine analysis using extensive sets of data allowed us to determine the appearance and retention of the genes-of-interest (GOIs) during evolution and the type of selection pressure they have been subject to. Parallelly, we have carried out a functional analysis of the candidate genes using reverse genetic in Arabidopsis thaliana. We produced plant lines allowing us to determine the time and place of our candidate genes expression and obtained a fine map of expression patterns. In addition, plants with mutated gene-of-interest have been obtained and genetically confirmed. For genes for which no mutant existed, we used CRISPR/Cas9 technics to obtain novel mutant alleles of the genes. The mutants were subject to a fine phenotypic analysis in different conditions to assess the impact of a non-functional gene on plant development. With this work, we selected a candidate gene showing subtle yet pleiotropic, hormone-like, phenotype when nonfunctional.
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.
At the beginning of the project very little was known regarding plant signaling compounds other that the well characterized phytohormones. We aimed at describing overlooked signaling compounds that are necessary to finely modulate plant growth. We proposed an approach using specific evolutionary signatures to select more potential players in signaling compounds biosynthesis and homeostasis. During this project we showed that it is possible to use genomic data publicly available to select cytochrome P450 genes showing specific evolutionary patterns and developed a streamlined method to categorized genes base on this approach. Of the several hundreds of cytochrome P450 in Arabidopsis thaliana, a large portion have no known functional data. During the course of this project, we obtained a fine map of expression patterns of 7 cytochrome P450 for which no functional data existed. We also obtained and confirm genetic tools, such as mutant alleles for our GOIs, and finely described phenotypic consequences of nonfunctional alleles (and sometimes overexpressors) on plant development when observed. During this project we particularly discovered and described one gene with significant impact on normal plant development and thus likely to be involved in an unknown signaling compound metabolism for the fine regulation of plant growth. More analyses are needed to confirm this hypothesis, but we believe that this cytochrome P450 might be of interest to understand an overlooked signaling pathways in seed plants and might be put to use for regulation of crop growth and tree development to optimize yield and cultural practices.
Cytochrome P450 structure
Candidate cytochrome P450's expression pattern