Vincent Chiang

The goal for this partnership is to plant, develop and document the information and tools needed to demonstrate the sustainable production of biomass for bioenergy across the Southern US. Specifically, this program will develop and demonstrate sustainable, flexible, integrated biomass production solutions that create innovative deployment scenarios to reliably produce and supply biomass feedstocks that are optimized for performance in leading conversion technologies. Research and development activities will target specific barriers in each step of the supply chain that are identified as critical to regional economic and/or environmental sustainability. Education, extension and outreach activities will be integrated so that the results of this work will reach target audiences with appropriate real-world examples.

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.

With the rapid increase in the world?s population, demand for wood as a material and as a fuel is expected to increase exponentially in the future. Biotechnology will play a major role in meeting this future wood demand. Traditional tree breeding has been proven effective in increasing forest productivity. Genetic engineering is also being used to produce trees that efficiently sequester carbon, are more amenable to treatment for biofuel production, and have desired wood properties. The tailoring of trees with desired characteristics for optimal processing offers a tremendous potential for the forest products industry in the United States. One factor that is crucial in this integration of biotechnology and manufacturing is the availability of individuals who have the skills of both a tree biologist and a materials engineer. The proposed fellowship?s goal is to address this issue. The project objective is to develop a training program to produce graduates with strong background in forest biotechnology, biometrics, and wood engineering. Two doctoral students will be trained in the Conservation and Renewable Natural Resources discipline (Code C) of the Forest Resources Targeted Expertise Shortage Area. One student will have an engineering background but will undergo expansive training in biometrics and forest biotechnology. The other student will have a forestry or biology background but will be provided additional training in the field of wood materials science and engineering. The fellows will start a new breed of scientists with interdisciplinary perspectives to tackle issues ranging from environmental sustainability to renewable energy.

The new equipment will serve a diverse user community. Because of its combined capability as a highthroughputb sequencer that generates reads ultra fast, it will be an essential tool in research requiring: 1) variant detection for genotyping breeding populations, 2) near real-time metagenomics remediation assays and other population surveys, 3) SNP detection, genotyping, and genetic mapping, 4) transcriptome sequencing and gene expression analysis, and 5) full genome and targeted resequencing (including ChIP seq and bisulfite-treated DNA applications)

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.

The account will be used for paying salaries for Vincent Chiang to administer and monitor all FORBIRC research activities and for research staff to work on and maintain facilities and equipment used by all FORBIRC projects, including materials and supplies and services associated with the maintenance. It will also cover all expenses associated with the annual FORBIRC meeting, including fees for meeting facilities, meals, etc. $3,000 will be used to construct the FORBIRC website for outreach and disseminating research knowledge and results associated with FORBIRC research projects

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.