Ron Sederoff

Plant cell walls are the essential components of feedstocks for biomass based liquid fuel alternatives to petroleum. The secondary cell walls of woody plants contribute greatly to biomass and are targets for improving potential feedstocks. In the application of systems biology to development of new biofuels, as in any complex biological process, predictive modeling is the central goal. We propose to use a systems approach with genome based information and mathematical modeling to advance the understanding of the biosynthesis of the plant secondary cell wall. To do this, we will use multiple transgenic perturbations and measure effects on plants using advanced quantitative methods of genomics, proteomics, and structural chemistry. The combination of quantitative analysis, transgenesis, statistical inference and systems modeling provide a novel and comprehensive strategy to investigate the regulation, biosynthesis and properties of the secondary cell wall.

Lignin is a unique and complex phenylpropanoid polymer, important in plant development and response to environment. We propose to advance our knowledge of lignin biosynthesis by developing a comprehensive pathway model of regulatory and metabolic flux control mechanisms. Our primary tool will be systematic gene specific perturbation in transgenic Populus trichocarpa. We will perturb all 34 known lignin pathway and regulatory network genes in P. trichocarpa using artificial microRNA (amiRNA) and RNAi suppression. From each independent transgenic perturbation, we will obtain quantitative information on transcript and protein abundance, enzyme kinetics, metabolite concentrations, and lignin structural chemistry. Using statistical correlation and path analysis, we will integrate this information to develop a mechanistic-based signaling graph and metabolic flux model for the pathway and its regulation leading to specific lignin structures. This model will reveal regulatory constraints on steady-state flux distributions and show how genes and other process components affect flux activity of lignin precursors, composition, and linkages. In this way, we will provide a systems biology approach to this fundamental pathway. There are few opportunities in higher plants to integrate genomics, biochemistry, chemistry and modeling to develop a comprehensive understanding of biosynthesis and structure of a major component of morphology and adaptation.

Angiosperm species, such as Eucalyptus globulus, have lignin composed of both syringyl and guaiacyl subunits. In contrast, gymnosperm species such as Pinus taeda have lignin which is devoid of syringyl monomer units. Favorable pulping efficiency with high pulp yields has long been associated to wood with lignin containing high syringyl component. Inducing the biosynthesis of syringyl subunits in gymnosperm lignin would be a direct approach to engineer gymnosperm wood for improved pulping efficiency and yields. Here, we propose to establish a strategy to demonstrate the induction of syringyl monolignol biosynthesis in gymnosperms through an in vitro protein activity assay system. In this system, we plan to spike the total protein mixtures isolated from the secondary differentiating xylem (SDX) of a gymnosperm with angiosperm syringyl-lignin specific recombinant proteins. We will assay these protein mixtures using coniferaldehyde, a common precursor to both guaiacyl and syringyl monolignols, and monitor the synthesis of sinapyl and coniferyl alcohol, precursors of syringyl and guaiacyl monolignol respectively. These assays will provide insights to the extent of syringyl monolignol biosynthesis we can induce into gymnosperms by the addition of angiosperm proteins.

WHEREAS, the parties to this Agreement intend to join together in a cooperative effort to support a FOREST BIOTECHNOLOGY INDUSTRIAL RESEARCH CONSORTIUM (hereinafter called ?CONSORTIUM?) at UNIVERSITY to develop a better understanding of fundamental information about the growth and development of trees, to promote innovative research for improved trees with desired properties using the most advanced forest biotechnology, to foster interactions between industry and university researchers, and to facilitate further research cooperation between the parties.

Whereas, the parties to this Agreement intend to join together in a cooperative effort to support a FOREST BIOTECHNOLOGY INDUSTRIAL RESEARCH CONSORTIUM (hereinafter called "CONSORTIUM") at UNIVERSITY to develop a better understanding of fundamental information about the growth and development of trees, to promote innovative research for improved trees with desired properties using the most advanced forest biotechnology, to foster interactions between industry and university researchers, and to facilitate further research cooperation between the parties.

In order to identify candidate genes involved in frost tolerance, we will establish a eucaluyptus differentiating xylem protoplasts system. We have established a protocol of protoplast isolation and highly efficient transfection for developing xylem of P. trichocarpa. We measured the accuracy of the P. trichocarpa xylem protoplast transient expression system using PtrSND1-B1, a transcription factor specific to the secondary cell wall biosynthesis. Our results suggest the xylem protoplasts system coupled with transcript profiling by RNA-seq provides robust analysis of expression induced by a specific introduced gene. We will use the clone BRASUIZ1 in this study. The genome of clone BRASUIZ1 has been sequenced, and will enable us to identify the genes that are exclusively expressed in the xylem by mapping the mRNA-seq data to the genomic database. We have successfully isolated the differentiating xylem protoplasts from eucaluyptus (Fig.1), and some adjustments for the procedures are required. We will focus on the identification of genes involved in frost tolerance, and also possible previously unidentified genes.

We will perform the whole transcriptome analysis of the differentiating xylem in Eucalyptus grandis clone BRASUIZ1. We will, through the analysis of the high-throughput RNA-seq data, identify the candidate genes associated with wood formation. Our focuses will be on the identification of the genes involved in cellulose, hemicelluloses, and lignin biosynthesis, and on the NAC and MYB family transcription factors for wood formation.

Laccases were proposed for lignin biosynthesis and for phenolic production. However, currently there are no reports of laccase functions on the monolignol polymerization in trees. We have identified 20 xylem-expressed laccases among the 79 laccase gene models in the P. trichcarpa genome. To study which laccases are involved in wood formation, we produced transgenics overexpressing Ptr-miR397a, which is predicted to target laccases. Our preliminary qRT-PCR showed gene expression reductions of selected six laccase genes, indicating successful functioning of Ptr-miR397a in the transgenic plants. We propose to characterize the transgenic plants for transcriptome and sugar composition analysis, including lignin content, lignin S/G ratio, and phenolic content. Correlation of the transcriptome and sugar composition changes will enable us to identify the laccases responsible for wood formation.

Whereas, the parties to this Agreement intend to join together in a cooperative effort to support a FOREST BIOTECHNOLOGY INDUSTRIAL RESEARCH CONSORTIUM (hereinafter called "CONSORTIUM") at UNIVERSITY to develop a better understanding of fundamental information about the growth and development of trees, to promote innovative research for improved trees with desired properties using the most advanced forest biotechnology, to foster interactions between industry and university researchers, and to facilitate further research cooperation between the parties.

Whereas, the parties to this Agreement intend to join together in a cooperative effort to support a FOREST BIOTECHNOLOGY INDUSTRIAL RESEARCH CONSORTIUM (hereinafter called "CONSORTIUM") at UNIVERSITY to develop a better understanding of fundamental information about the growth and development of trees, to promote innovative research for improved trees with desired properties using the most advanced forest biotechnology, to foster interactions between industry and university researchers, and to facilitate further research cooperation between the parties.