Lignin is a significant recalcitrant in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. We identified candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon and developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1-silenced and wildtype plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4-downregulated plants exhibited a decrease in total lignin content, a significant reduction of guaiacyl lignin, and a modest reduction of syringyl lignin. Ethanol yield by microbial fermentation was enhanced in both BdCAD1- and BdCOMT4-downregulated plants. These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater biomass yield and bioconversion efficiency.

Transcriptomics and gene expression profiling enables the elucidation of the genetic response of an organism to various environmental cues. Transcriptomics enables the deciphering of differences between two closely related organisms to the same environment and in contrast, enables the elucidation of genetic responses of the same organism to different environmental cues. Two major methods are utilized for the study of transcriptomes, high-throughput sequencing and microarray analysis. High-throughput sequencing technologies such as the Illumina platform are relatively new and protocols must be developed for the analyses of transcriptomes (RNA-sequencing). A RNA-seq protocol was developed and refined for the Illumina sequencing platform. This protocol was then utilized for the de novo sequencing of the steelhead salmon transcriptome. Hatchery steelhead exhibit a reduced fitness compared to wild steelhead that has been shown to be genetically based. Consequently, the steelhead transcriptome was assembled, annotated, and used to identify gene expression differences between hatchery and wild fish. We uncovered many differentially expressed genes involved in metabolic processes and growth and development. This work has created a better understanding of the genetic differences between hatchery and wild steelhead salmon. Brachypodium distachyon is a monocot grass important as a model for cereal crops and potential biofuels feedstocks. To better understand the genetic response of this plant to different environmental cues, a comprehensive assessment of the transcriptomic response was conducted under a variety of conditions including diurnal/circadian light/dark/temperature environments and different abiotic stress conditions. Using a whole-genome tiling DNA microarray, we identified that the majority of transcripts in Brachypodium exhibit a daily rhythm in their abundance that is conserved between rice and Brachypodium. We also identified numerous cis-regulatory elements dictating these rhythmic expression patterns. We also identified the genetic response to abiotic stresses such as salinity, drought, cold, heat, and high light. We uncovered a core set of genes which responds to all stresses, indicating a core stress response. A large number of transcription factors were uncovered as potential nodes for regulating the abiotic stress response in Brachypodium. Moreover, promoter elements that drive specific responses to discrete abiotic stresses were uncovered. Altogether, the transcriptome analyses in this work furthers our understandings of how particular organisms respond to environmental cues and better elucidates the relationship between genes and the environment. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Oct. 5, 2011 - April 5, 2012.

La récente définition de Brachypodium distachyon comme modèle des graminées en fait un organisme de choix pour l'étude de leur paroi cellulaire, en particulier dans le cadre de leur utilisation comme matière première renouvelable pour le bioéthanol de seconde génération. Les lignines, dont les trois unités (H, G et S) proviennent de la polymérisation des monolignols, sont associées aux acides hydroxycinnamiques dans la paroi des céréales et représentent l'obstacle majeur à l'exploitation industrielle de la biomasse lignocellulosique. L'acquisition de connaissances sur les mécanismes dirigeant leur mise en place et leur organisation permettrait d'identifier des facteurs modulant les rendements de production qui y sont associés. Quatre familles de gènes ont été étudiées et l'implication dans la voie de biosynthèse des monolignols de trois gènes a été montrée : BdF5H2 possède une activité férulate-5-hydroxylase permettant la synthèse des précurseurs des unités S des lignines, BdCOMT3 est l'isoforme principale des acide cafféique O-Méthyltransférases et sa perte partielle de fonction cause une diminution de la quantité de lignine, la modification du rapport S/G et une baisse de quantité d'acide p-coumarique dans deux lignées mutantes indépendantes. Enfin, BdCAD1 est l'isoforme principale des alcools cinnamylique déshydrogénases : sa perte de fonction dans deux lignées indépendantes cause la diminution de la quantité globale de lignine et d'acide p-coumarique, une baisse du rapport S/G ainsi que l'accumulation de sinapaldéhyde. Par ailleurs ces deux lignées présentent des rendements de saccharification augmentés de plus d'un quart par rapport au sauvage.

Plants reduce inorganic carbon to synthesize biomass that is comprised of mostly polysaccharides and lignin. Growth is intricately regulated by external cues such as light, temperature, and water availability and internal cues including those generated by the circadian clock. While many aspects of polymer biosynthesis are known, their regulation and distribution within the stem are poorly understood. Plant biomass is perhaps the most abundant organic substance on Earth and can be used as feedstock for energy production. Various grass species are under development as energy crops yet several of their attributes make them challenging research subjects. Brachypodium distachyon has emerged as a grass model for food and energy crop research. I studied rhythmic growth, a phenomenon important to understanding how plant biomass accumulates through time, and vascular system development, which has biofuel feedstock conversion efficiency and yield. Growth rate changes within the course of a day in a sinusoidal fashion with a period of approximately 24 hours, a phenomenon known as rhythmic growth. Light and temperature cycles, and the circadian clock determine growth rate and the timing of rate changes. I examined the influences of these factors on growth patterns in B. distachyon using time-lapse photography. Temperature and, to a lesser extent, light influenced growth rate while the circadian clock had no noticeable effect. The vascular system transports important materials throughout the plant and consists of phloem, which conducts photosynthates, and xylem, which conducts water and nutrients. The cell walls of xylem elements and ground tissue sclerenchyma fibers are comprised of cellulose, hemicelluloses, and lignin. These components are important to alternative energy research since cellulose and hemicellulose can be converted to liquid fuel, but lignin is a significant inhibitor of this process. I investigated vascular development of B. distachyon by applying various histological stains to stems from three key developmental. My results described in detail internal stem anatomy and demonstrated that lignification continues after crystalline cellulose deposition ceases. A better understanding of growth cues and various anatomical and cell wall construction features of B. distachyon will further our understanding of plant biomass accumulation processes.