Project Summary: Biosynthesis and Genetic Manipulation of Lignin

Biosynthesis and genetic manipulation of lignin

Project Objectives:
The aim of this project is to determine the pathways leading to the biosynthesis of the monolignol building blocks of lignin in the forage legume alfalfa. Approaches include transgenic down-regulation of key enzymes in the pathway, coupled to detailed analysis of corresponding changes in lignin content and composition, and global metabolite profiles. Genomics approaches using DNA microarray analysis are being used to monitor changes in gene expression associated with down-regulation of the different target genes, or as a result of altered lignin/cell wall composition. Through collaboration with Dr. John Ralph, US Forage and Dairy Research Center, Madison, WI, various NMR techniques are being applied to provide a detailed picture of the lignin structure in the various transgenic lines generated. We are interacting with Dr. Lloyd Sumner's group at Noble to study the effects of genetic modification on the cell wall proteome. Through collaboration with Dr. Joe Noel of the Salk Institute Structural Biology Laboratory, we are investigating the potential for structure-based modification of enzymes to provide novel strategies for the genetic modification of lignin.

Present Lab Personnel on Project:

Past Personnel:

Dr. Jin Nakashima
Dr. Srinu Reddy
Dr. Dianjing Guo
Dr. Ganesan Gowri
Dr. Kentaro Inoue
Dr. Parvathi Kota
Dr. Weiting Ni
Dr. Vincent Sewalt

Collaborators:

Dr. Joe Noel, Salk Institute Structural Biology Laboratory, San Diego, CA
Dr. John Ralph, US Forage and Dairy Research Center, Madison, WI
Dr. Lloyd Sumner, Noble Foundation
Dr. Stephen Temple, Forage Genetics International, Wets Salem, WI

Sources of Funding:

Noble Foundation
Forage Genetics International
US Department of Energy

Recent Publications:

Ralph, J., Brunow, G., Harris, P., Dixon, R.A. and Boerjan, W. (2008). Lignification: are lignins biosynthesized via simple combinatorial chemistry or via proteinaceous control and template replication? Recent Advances in Polyphenols Research, 1, in press.
Nakashima, J., Chen, F., Jackson, L., Shadle, G., and Dixon, R.A. (2008). Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa L.) - effects on lignin composition in specific cell types. New Phytologist 179, 738-750.
Jackson, L.A., Shadle, G.L., Zhou, R., Nakashima, J., Chen, F., and Dixon, R.A. (2008). Improving saccharification efficiency of alfalfa stems through modification of the terminal stages of monolignol biosynthesis. BioEnergy Research 1, 180-192.
Shadle, G., Chen, F., Reddy, M.S.S., Jackson, L., Nakashima, J., and Dixon, R.A. (2007). Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxy cinnamoyl transferase in transgenic alfalfa impacts lignification, development and forage quality. Phytochemistry 68, 1521-1529.
Chen, F., and Dixon, R.A. (2007) Lignin modification improves fermentable sugar yields for biofuel production. Nature Biotechnology 25, 759-761.
Ralph, J., Akiyama, T., Kim, H., Lu, F., Schatz, P.F., Marita, J.M., Ralph, S.A., Reddy, M.S.S., Chen, F. and Dixon, R.A. (2006) Effects of coumarate 3-hydroxylase down-regulation on lignin structure. Journal of Biological Chemistry 281, 8843-8853.
Reddy, M.S.S., Chen, F., Shadle, G., Jackson, L., Aljoe, H. and Dixon, R.A. (2005). Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa (Medicago sativa L.). Proceedings of the National Academy of Sciences USA 102 16573-16578.
Ferrer, J.L., Zubieta, C., Dixon, R.A. and Noel, J.P. (2005). Crystal structures of alfalfa caffeoyl CoA 3-O-methyltransferase. Plant Physiology, in press.
Kota, P., Guo, D., Zubieta, C., Noel, J. and Dixon, R.A. (2004). O-Methylation of benzaldehyde derivatives by "lignin specific" caffeic acid 3-O-methyltransferase. Phytochemistry, 65, 837-846.
Dixon, R.A. (2004). Molecular improvement of forages- from genomics to GMOs. In Molecular Breeding of Forage and Turf, eds A. Hopkins, Z-Y Wang, R. Mian, M. Sledge and R.E. Barker, Kluwer Academic Publishers, 1-19.
Dixon, R.A. and Reddy, M.S.S. (2003). Biosynthesis of monolignols. Genomic and reverse genetic approaches. Phytochemical Reviews 2, 289-306.
Chen, L., Auh, C.-K., Dowling, P., Bell, J., Chen, F., Hopkins, A., Dixon, R.A. and Wang, Z.-Y. (2003) Improved forgage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase. Plant Biotechnology Journal 1, 437-449.
Chen, F., Duran, A.L., Blount, J.W., Sumner. L.W. and Dixon., R.A. (2003). Profiling phenolic metabolites in transgenic alfalfa modified in lignin biosynthesis. Phytochemistry, 64, 1013-1021.
Marita, J.M., Ralph, J.R., Hatfield, R.D., Guo, D., Chen, F., and Dixon, R.A. (2003). Strucutral and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase. Phytochemistry 62, 53-65.
Chen, L., Auh, C., Chen, F., Cheng, X. F., Aljoe, H., Dixon, R. A. and Wang, Z. Y. (2002) Lignin deposition and associated changes in anatomy, enzyme activity, gene expression and ruminal degradability in stems of tall fescue at different developmental stages. Journal of Agricultural and Food Chemistry, 50, 5558-5565.
Guo, D., Chen, F. and Dixon, R.A. (2002) Monolignol biosynthesis in microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.) Phytochemistry 61, 657-668.
Zubieta, C., Kota, P., Ferrer, J-L., Dixon, R.A. and Noel, J. (2002). Structural basis for the modulation of lignin monomer methylation by caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase. Plant Cell 14: 1265-1277.
Chen, F., Parvathi, K., Blount, J.W. and Dixon, R.A. (2001). Chemical syntheses of caffeoyl and 5-hydroxyconiferyl aldehydes and alcohols for determination of lignin O-methyltransferase activities in dicot and monocot species. Phytochemistry 58: 1035-1042.
Guo, D., Chen, F., Wheeler, J., Winder, J., Selman, S., Peterson, M. and Dixon, R.A. (2001). Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases. Transgenic Research 10: 457-464.
Dixon, R.A., Chen, F., Guo, D. and Parvathi, K. (2001). The biosynthesis of monolignols: a "metabolic grid", or independent pathways to guaiacyl and syringyl units? Phytochemistry 57: 1069-1084.
Dixon, R.A., Chen, F., Guo, D., He, X.-Z., Liu, C.-J., Noel, J.P., Parvathi, K., and Zubieta, C. (2001). Properties and metabolic engineering of alfalfa O-methyltransferases of phenylpropanoid biosynthesis. Recent Advances in Phytochemistry 35: 131-151.
Parvathi, K., Chen, F., Guo, D., Blount, J.W. and Dixon, R.A. (2001). Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols. Plant Journal 25: 93-102.
Guo, D., Chen, F., Blount, J., Inoue, K. and Dixon, R.A. (2001). Down-regulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa (Medicago sativa L.). Effects on lignin structure and implications for the biosynthesis of guaiacyl and syringyl lignin. Plant Cell 13: 73-88.
Inoue, K. and Dixon, R.A. (2000) Substrate preferences of caffeic acid/ 5-hydroxyferulic acid 3-O-methyltransferases in developing stems of alfalfa (Medicago sativa L.). Archives of Biochemistry and Biophysics 375: 175-182.