DOWNREGULATION OF CINNAMOYL-COENZYME A REDUCTASE IN POPLAR: MULTIPLE-LEVEL PHENOTYPING REVEALS EFFECTS ON CELL WALL POLYMER METABOLISM AND STRUCTURE

Authors: LEPLE, JEAN-CHARLES - INRA-ARDON; DAUWE, REBECCA - VIB, GENT, BELGIUM; MORREEL, KRIS - VIB, GENT, BELGIUM; STORME, VERONIQUE - VIB, GENT, BELGIUM; LAPIERRE, CATHERINE - CTP, GRENOBLE, FRANCE; POLLET, BRIGITTE - CTP, GRENOBLE, FRANCE; NAUMANN, ANNETTE - UNIV GOTTINGEN, GERMANY; KANG, KYU-YOUNG - UNIV OF BRITISH COLUMBIA; KIM, HOON - UNIV OF WISCONSIN; RUEL, KATIA - CTP, GRENOBLE, FRANCE; LEFEBVRE, ANDREE - CTP, GRENOBLE, FRANCE; JOSSELEAU, JEAN-PAUL - CTP, GRENOBLE, FRANCE; GRIMA-PETTENATI, JACQUELINE - UMR, TOULOUSE, FRANCE; DE RYCKE, RIET - GENT UNIV, GENT, BELGIU; ANDERSSON-GUNNERAS, SARA - SWEDISH UNIV AG SCI, UMEA; ERBAN, ALEXANDER - MAX-PLANCK INST, GERMANY; FEHRLE, INES - MAX-PLANCK INST, GERMANY; PETIT-CONIL, MICHEL - CTP, GRENOBLE, FRANCE; KOPKA, JOACHIM - MAX-PLANCK INST, GERMANY; POLLE, ANDREA - UNIV GOTTINGEN, GERMANY; MESSENS, ERIC - VIB, GENT, BELGIUM; SUNDBERG, BJORN - UMEA, SWEDEN; MANSFIELD, SHAWN - UNIV BRIT COLUMBIA,CANAD; Ralph, John; PILATE, GILLES - INST NAT RECHER AGRON, FR; BOERJAN, WOUT - GENT UNIV, GENT, BELGIUM

Submitted to: The Plant Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/16/2007
Publication Date: 11/1/2007
Citation: Leple, J., Dauwe, R., Morreel, K., Storme, V., Lapierre, C., Pollet, B., Naumann, A., Kang, K., Kim, H., Ruel, K., Lefebvre, A., Josseleau, J., Grima-Pettenati, J., De Rycke, R., Andersson-Gunneras, S., Erban, A., Fehrle, I., Petit-Conil, M., Kopka, J., Polle, A., Messens, E., Sundberg, B., Mansfield, S.D., Ralph, J., Pilate, G., Boerjan, W. 2007. Downregulation of cinnamoyl-coenzyme a reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure. The Plant Cell. 19:3669-3691.

Interpretive Summary: CCR is an enzyme used by plants in the production of monolignols, the building blocks from which the polymer lignin is produced. (It is lignin that holds fibers together in woody and forage plants; lignin is essential for the plant, but is a limit to plant cell wall digestibility by ruminant animals and an impediment in industrial pulping to make paper, or bioconversion to alternative fuels. Consequently there is a lot of effort directed toward selection and genetic methods for altering lignins). Here, we report on the generation of transgenic poplar trees downregulated for CCR and the molecular and growth characteristics of these transformed plants. CCR downregulation was associated with up to 50% reduced lignin content and an orange-brown, often patchy coloration of the outer wood. We showed that the coloration reflects the incorporation of ferulic acid in the lignin of the transformants; ferulic acid is not normally considered a lignin monomer. Its incorporation gives rise to a characteristic structure that efficiently releases a diagnostic marker compound following thioacidolysis, a useful analytical method for lignins. The cohesion of the cell walls was affected. Both lignin and non-cellulose polysaccharides could be more easily extracted from the transformed plants by pulping. An altered xylan metabolism was observed; xylan, like cellulose, is a major class of polysaccharides in these plants. Data pointed toward a stress response induced by the altered cell wall structure. The improved pulping characteristics of field-grown transformed plants indicated the potential economic value of CCR suppression if reduced growth effects can be addressed. These and other studies are ultimately aimed at enhancing the utilization of valuable plant cell wall resources in a variety of processes.

Technical Abstract: Cinnamoyl-CoA reductase catalyzes the penultimate step in monolignol biosynthesis. Here, we report on the generation of transgenic poplars down-regulated for CCR and the molecular and growth characteristics of these transformants. CCR down-regulation was associated with up to 50% reduced lignin content and an orange-brown, often patchy coloration of the outer xylem. We showed that the coloration reflects an increased incorporation of ferulic acid in the lignin of the transformants. Its incorporation gives rise to a characteristic substructure that efficiently releases a dianiostic momomer following thioacidolysis. Elevated ferulic acid levels were observed in the form of esters and oxidatively copolymerized into the lignin. The cohesion of the walls was affected, particularly at sites that are rich in S-lignin units in WT but depleted in S-units in the transformants. Both lignin and hemicellulose could be more easily extracted from the transformants as compared to WT, by Kraft pulping and alkali treatment. Furthermore, an altered xylan metabolism was observed based on transcript and metabolite profiling and confirmation by FTIR. Transcript levels of two PAL genes were induced in the transformants and, in general, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure, resulting in the induction of carbon flux toward ferulic acid biosynthesis, for the reinforcement of the damaged wall. The improved pulping characteristics of field-grown transformants indicated the potential economic value of CCR suppression. Growth indicators were however affected for all transgenic lines tested.