Metabolic analysis of Arabidopsis plants with altered lignification

Lignin is, after cellulose, the second most abundant terrestrial biopolymer, accounting for approximately 30% of the organic carbon in the biosphere. The ability to synthesize lignin has been essential in the evolutionary adaptation of plants from an aquatic environment to land. Lignin is crucial for structural integrity of the cell wall and stiffness and strength of the stem. In addition, lignin waterproofs the cell wall, enabling transport of water and solutes through the vascular system, and plays a role in protecting plants against pathogens. Lignin is an aromatic heteropolymer, abundantly present in the walls of secondary thickened cells. Lignins are complex natural polymers resulting from oxidative coupling of, primarily, 4-hydroxyphenylpropanoids. An understanding of their nature is evolving as a result of detailed structural studies, recently aided by the availability of lignin-biosynthetic-pathway mutants and transgenics. The currently accepted theory is that the lignin polymer is formed by combinatorial-like phenolic coupling reactions, via radicals generated by peroxidase-H2O2, under simple chemical control where monolignols react endwise with the growing polymer. As a result, the actual structure of the lignin macromolecule is not absolutely defined or determined. The “randomness” of linkage generation (which is not truly statistically random but governed, as is any chemical reaction, by the supply of reactants, the matrix, etc.) and the astronomical number of possible isomers of even a simple polymer structure, suggest a low probability of two lignin macromolecules being identical. A recent challenge to the currently accepted theory of chemically controlled lignification, attempting to bring lignin into line with more organized biopolymers such as proteins, is logically inconsistent with the most basic details of lignin structure. Lignins may derive in part from monomers and conjugates other than the three primary monolignols (p-coumaryl, coniferyl, and sinapyl alcohols). The plasticity of the combinatorial polymerization reactions allows monomer substitution and significant variations in final structure which, in many cases, the plant appears to tolerate. We have profiled the methanol-soluble oligolignol fraction of Arabidopsis thaliana stem, a tissue with extensive lignification. Two genes have been modified to study the variation in lignin composition, F5H and COMT, respectively up and downregulated. At the beginning, we deeply worked on a new liquid chromatography machine (UPLCTM) optimizing the several parameters which permit an optimal separation of the molecules. This operation took us a lot of time but these technical parameters are now used by the research group. Using liquid chromatography-mass spectrometry, we tried to discover new monomers being generated from the reshape monomer pathway. Although we could not present any new molecule, we had experience about machine settings and laboratory pratical skills. To describe the practical aspects of my work, in this thesis I am presenting a research on poplar lignins made by the same research group I worked with