Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types.

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types.

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types. / Science, Faculty of / Botany, Department of / Graduate