Revealing complexity of the plant phenolic metabolism : novel routes to the precursors of the cell-wall biopolymers – PHENOWALL

Target enzymes were expressed in microorganisms to determine their respective specific activities. Their expression profiles in the plant were defined. The role of each of them in the plant has been determined using knock-down and overexpression mutants, which have been analysed for their developmental phenotype and for soluble phenolics and lignin composition. Instrumental to this work were the recently implemented mutants collections at IGPB and in USA.

Results of PHENOWALL demonstrate that the duplication of the second enzyme of the phenolic metabolism during plant evolution lead to a functional diversification without modification of the principal enzyme activity: hydroxylation of cinnamic acid is carried out with similar efficiencies by the different duplicates (paralogs) in spite of significant differences in the structure of their active sites. The subcellular localization of the different paralogs expressed in a heterologous system (leaf epidermal cells) appears to be the same (endoplasmic reticulum membranes). Our results however show that their anchoring mode in the membranes is different and leads to a differential membrane topology. Moreover, one of the paralogs was found to be glycosylated. Analysis of their expression profiles during plant development shows that some of them are expressed in roots, reproductive organs, or are associated to specific cell types. Suppression and overexpression mutant analysis is still on-going. Preliminary results indicate either a moderate impact on plant development and lignin composition of the individual suppression of target genes, or a suppression of plant viability. The production of double mutants is under way to overcome functional redundancies. Mutant analysis carried-out in the project also revealed the function of other genes involved in the plant lignification
PHENOWALL also provided the basis and tools for the initiation of other collaboration with Canada (the Working On Wood project), Belgium, Germany and USA.
New projects initiated on the basis of PHENOWALL:
- METABEVO project: USIAS-FRIAS joint project with Freiburg University on phenolic metabolism evolution in moss 2013-2015 (partner 1)
- PEACH project: submitted to Institut Carnot 3BCAR on the production of férulic acid as high added value compound in Brachypodium
- BRAVO project: funded by ANR on the ontogeny of the vascular tissues in Brachypodium.

The PHENOWALL project will identify markers for modifying the phenolic composition and reticulation of lignin, in order to improve production of biofuels from cereals straws and stovers, and to improve forage digestibility. It should also contribute to reveal how to optimize phenolic composition of flowers and seeds/fruits. Preliminary data suggest impacts on plant development, flowering time and fertility. These data however require further validation.

Complete annotation of the cytochrome P450 genes from Brachypodium and discussion on evolution of the P450 family: Nelson D, Werck-Reichhart D. (2011) A P450-centric view of plant evolution. Plant J. 66:194-211.

New approaches for the analysis of the protein-protein interactions in the phenolic metabolism: Bassard JE, Richert L, Geerinck J, Renault H, Duval F, Ullmann P, Schmitt M, Meyer E, Mutterer J, Boerjan W, De Jaeger G, Mely Y, Goossens A, Werck-Reichhart D. (2012) Protein-Protein and Protein-Membrane Associations in the Lignin Pathway. Plant Cell 24:4465-4482.

Publication of the TILLING of the P450 genes: Dalmais M, Antelme S, Ho-Yue-Kuang S, Wang Y, Darracq O, Bouvier d’Yvoire M, Cézard L, Légée F, Blondet E, Oria N, Troadec C, Brunaud V, Jouanin L, Höfte H, Bendahmane A, Lapierre C, Sibout R (2013). A TILLING Platform for Functional Genomics in Brachypodium distachyon. Plos One. 8: e65503.

Discovery of the first mutant in a Poaceae with lost p-coumaryl monolignol transferase function: Petrik DL, Karlen SD, Cass CL, Padmakshan D, Lu F, Liu S, Le Bris P, Antelme S, Santoro N, Wilkerson CG, Sibout R, Lapierre C, Ralph J, Sedbrook JC (2014). p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon. Plant J. 77:713-26.

The plant phenolic metabolism converts phenylalanine into building blocks of cell-wall biopolymers (lignin, suberin, sporopollenin, cross-linkers) and into a large diversity of abundant soluble compounds (phenolic esters, phenolamides, flavonoids, coumarins, etc) with antioxydant or organoleptic properties. The pathways leading to phenolic compounds are still not completely understood despite their economical importance and extensive genetic engineering recently undertaken for improving biomass saccharification, paper production or nutritional qualities of the plants. In fact, biochemical analysis of genetically engineered plants suggests that some branches in the pathway have been overlooked so far. The aim of PHENOWALL is to reveal some of these branch pathways, using as reporters the cytochrome P450 hydroxylases of the phenolic ring.
The CYP73 and CYP98 families of P450 enzymes have been reported to catalyze the 4- and 3-hydroxylations of the phenolic rings. CYP73s accept free cinnamic acid as a substrate. Recent data suggest that CYP98s catalyze meta-hydroxylation of a range of hydroxycinnamic esters and amides, some with broad specificity, while others narrowed their substrate selectivity. Phylogenetic reconstructions indicate a major and early duplication in each gene family both in monocots and dicots, prior to their divergence in the case of CYP73s. Early divergences are indicative of the acquisition of specific functions, most likely in different branches of the phenolic pathway. We thus propose to investigate the specific biochemical and biological functions of the CYP73 and CYP98 paralogues using in parallel screening of recombinant enzyme catalytic activities and functional analysis using the grass Brachypodium distachyon as a model. Brachypodium is a close relative of the temperate grass crops such as wheat and barley, and a good model for second generation biofuel crops. Its stature and generation time are compatible with lab work. This plant is now the focus of international efforts to establish it as a genetic model, including sequencing and mutant collections.
Our workplan includes the annotation and phylogeny of the CYP73 and CYP98 genes in the Brachypodium genome for identification of the members of each clan. Relevant candidates will then be extensively characterized for catalytic activity of the recombinant enzymes expressed in yeast. This work will be supported by the synthesis of a broad set of candidate substrates representative of the phenolic derivatives present in the plant and by homology modeling of the proteins for prediction of the active site divergence and docking of the hit compounds. In parallel, functional characterization will be carried out by 1) a detailed description of the expression patterns of the different paralogues via transcriptomics, q-PCR and promoter::GUS constructs visualization, 2) isolation of a set of TILLING mutants for each candidate and full analysis of gene impact on plant fertility and development, biopolymers and soluble phenolic composition.
This work will be carried out by a multidisciplinary consortium assembling competencies in enzymology and functional genomic of cytochromes P450, Brachypodium genetics and lignin/cell-wall analysis, synthetic chemistry, bioinformatics and structural biology in order to provide an extensive picture of the phylogeny/expression/structure/function (catalytic and biological) relationship of the different CYP73 and CYP98 hydroxylases. This investigation should reveal additional complexity in the phenolic metabolism with potential applications in plant improvement for biofuel and paper production, and nutritional and organoleptic value. It should provide novel routes to obtain building blocks for natural and artificial polymers.