Regional Competitive Grants

Project Title:  A novel approach to facilitate accessibility of cellulose and hemicellulose: characterization of hybrid poplar transformed with a tyrosine rich peptide gene

Principal Investigators and affiliations:

•  PI: Haiying Liang (Clemson University, Department of Genetics and Biochemistry)

 Start Date: 9/1/2007      End Date: 8/31/2008

Project Abstract:

Due to the importance of lignin in limiting enzymatic access to cellulose and hemicelluloses, investigators have attempted to genetically engineer trees to modify the lignin content /monomer composition. However, it is challenging to alter lignin content or composition without compromising tree health. Research thus far has provided mixed results. Rather than altering total lignin content or lignin monomer ratios, we propose to modify lignin structure by introducing new peptide cross-links. Our hypothesis is that replacing a small fraction of lignin-lignin linkages with lignin-peptide linkages will not compromise plant fitness but will facilitate the “cracking” of lignin so that the cellulosic components can be more easily hydrolyzed. The proposed approach is to introduce peptides/proteins containing a high tyrosine content into the cell walls of lignifying plant tissues where the peptidyl tyrosines will provide reactive sites for phenolic cross-links with lignin. The goal of the research is to develop trees with a lignin polymer that exhibits little or no change in lignin content and structure, but that could be easily removed by the action of proteases. A total of 24 hybrid poplar transgenic lines have been generated, containing a tyrosine-rich peptide gene. Preliminary data indicated that introduction of the transgene did not change the lignin content and morphology in stems. However, the effects of transgene product on lignin digestibility and wood tensile strength remained to be demonstrated. The specific objectives of the proposed study include: 1:Characterization of the transgenic plants for fitness - including lignin content , localization of the Tyr-rich peptides. Transformed plants will be compared to wild type in terms of growth rate, form, disease resistance, wood production, and basic wood physical properties; 2:Testing the lignin extractability in wood chips from transgenic plants with protease treatment.

Our hypothesis is that by replacing even a small fraction of lignin-lignin linkages with peptide linkages, we will not compromise plant fitness but will facilitate the “cracking” of lignin such that the cellulosic components are more easily hydrolyzed. This will be accomplished by introducing tyrosine-rich peptides into the cell walls of lignifying plant tissues via genetic transformation. It has been demonstrated that proteins naturally embed in the lignin of cell walls during development. This proximity should facilitate greater cross-linking, particularly in cases where tyrosine phenolic groups are prevalent. Our strategy to increase protein cross linking in lignifying tissue by transformation with additional tyrosine-rich proteins will only work if the wild-type levels of tyrosine-rich proteins in poplar cell walls are not already at saturation levels. Also, some proteins, such as are arabinogalactans and extensins, are secreted into the plant cell wall and thought to have specific functions necessary for plant growth. If higher levels of tyrosine-rich proteins in the cell wall displace other necessary proteins, then unsatisfactory phenotypes would be obtained in the transgenic plants. Not enough is yet known about the role of proteins in cell wall structure to be able to predict such pleiotropic effects. One benefit of the proposed transgenic study will be to obtain such critically needed knowledge about the structure of plant cell walls. Another possible pitfall is the ability of the laccases or peroxidases to cross-link the tyrosine phenols with the phenylpropanoid precursors. The lack of reactivity would decrease the extent of cross-linking. Nevertheless, we believe that these possible pitfalls, where the phenotype of the mature plant cannot be predicted, are in line with many of the other approached taken to alter lignin biosynthesis. In regard to the characterization studies, our approaches have a well-supported base in the literature.

Reports and Publications:

Quarter 1 - 2008
Quarter 2 - 2008
Quarters 3-4 - 2008
Project Final Report

Improved Sugar Release from Lignocellulosic Material by Introducing a Tyrosine-rich Cell Wall Peptide Gene in Poplar - Clean Air, Soil, and Water 2008, 36(8). 662-668