Objectives and Approaches

 
Our primary objective is to quantitatively describe how the flux and direction of the entire pathway for the biosynthesis of the lignin polymer is integrated and regulated. Our primary tool is systematic gene specific perturbation in transgenic Populus trichocarpa. We have identified, in the P. trichocarpa genome, all known pathway (22 monolignol and 4 PO) and transcription factor (2 LIM, 4 MYB, 1 KNOX) genes and their corresponding proteins (using LC-tandem mass spectrometry (MS/MS)), affecting lignin biosynthesis in differentiating xylem. We will perturb all these 33 genes using artificial microRNA (amiRNA) and RNAi suppression. We will:
 
(1) Quantify the responses (transcripts, proteins, metabolites, lignin composition and linkages) for every relevant gene through xylem-specific and gene-specific knock-down.
 
(2) Quantify redundancy where more than one member of a gene family is expressed using gene and family specific suppression.
 
(3) Generate new hypotheses about mechanisms of regulation and metabolic flux from data summaries and statistical analyses.
 
(4) Model and illustrate how changes in processing components affect pathway flux and lignin structure.
 
Consequently, we also expect to reveal new regulatory mechanisms and to answer the following questions: To what extent can the relative abundance of transcripts of specific genes predict the protein quantity? To what extent can the abundance and activity of individual enzymes predict the composition of lignin monomers? To what extent can the relative abundance of lignin monomers predict the quantity, composition, and specific linkages of lignin? Our long term goal is a predictive model of lignin biosynthesis and structure.
 
Specific Objectives:
 
1. Transgene Perturbation: We will generate transgenic P. trichocarpa with modified expression of all identified lignin pathway and TF genes.
 
2. Transcriptome Analysis: Transgenics for each gene will be analyzed to test for specificity and pleiotropic effects on transcripts within and outside of the lignin pathway using microarrays, qRT-PCR, and targeted sequencing.
 
3. Proteomic Analysis: Changes in abundance of protein components will be characterized by protein cleavage coupled with isotope dilution MS (PC-IDMS)-based LC-MS/MS for absolute protein quantity, and by LC-MS for enzyme activity. Kinetic parameters will be determined for recombinant proteins from all lignin biosynthesis genes.
 
4. Metabolite Quantitation: GC-MS and LC-MS/MS, coupled with stable isotope-labeled metabolites as internal standards (stable isotope dilution technique), will be used to quantify the effect of transgenic perturbations on type and concentration of lignin pathway metabolites to correlate metabolites and lignin structure.
 
5. Lignin Quantity and Structure: NIR will be used to quantify lignin content of the wood for each transgenic line. Lignin monomer composition and inter-unit linkages will be quantified by 1D and 2D NMR to identify correlations with pathway components and for modeling formation of lignin structures.
 
6. Database and Website: A public database/website will be setup at the beginning of the project to integrate all project data and information, including a comprehensive NMR structure library for lignin.
 
7. Statistical Analysis: Univariate and multivariate statistical methods will be applied to describe the degree, direction and significance of relationships among all pathway components and inter-unit linkages. Statistics is essential to establish significance of correlations and to provide quantitative information to mechanistic modeling.
 
8. Mechanistic and Systems Modeling: Modeling techniques will integrate experimental results and statistical inference to develop mathematical representations of lignin biosynthesis and linkage structure. We will construct a signaling graph and integrate the information into steady-state regulatory-constrained flux balance analysis (RC-FBA).
 
Detailed experimental approaches to accomplish the specific objectives are described in our project website (LigninSystems.org). All information and data generated in this project, as they are collected and analyzed, will be posted on this website. All biological materials, including transgenic plants, will also be made available following policies of NSF Plant Genome Program funded research.