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Perkins, Mendel L.

University of British Columbia

Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. The question of how lignin precursors (monolignols) move from inside the cells, where they are synthesized, across the cell membrane to the cell walls, where they are polymerized by laccase and peroxidase enzymes, has remained unanswered. Computer modelling indicates that monolignols could passively diffuse through lipid bilayer membranes, however, this has not been studied experimentally. This study tests the hypothesis that monolignol diffusion occurs when monolignols in the cell wall are consumed by lignin polymerization, due to the activity of oxidative enzymes like laccases and peroxidases. Liposomes containing monolignol-polymerizing laccase enzymes were developed as a model system to test monolignol diffusion across synthetic lipid bilayers. Not only did diffusion across the membrane occur, but lignin-like polymers formed within the liposomes. In parallel, two-photon microscopy of lignifying Arabidopsis root xylem vessels was used to visualize lignin polymer in the cell wall and soluble phenolics inside cells. Loss of function laccase mutants had monolignols accumulation in the vacuoles of neighbouring cells as detoxified monolignol glucosides, consistent with a diffusion-based transport (Chapter 2). The interaction of monolignol production and consumption was studied by using plants with upregulation in monolignol synthesis and overexpression of laccases. Monolignol over-producing lines were severely dwarfed, while the addition of laccase in the cell walls restored wild type growth and metabolic status. This suggests that the balance of monolignol production and consumption is finely controlled and has wide reaching transcriptional and developmental consequences when perturbed (Chapter 3). When lignin monomers were over-produced or left unpolymerized, increased monolignol glucosides stored in the vacuole were observed, and this was correlated with increased transcription of ATP binding cassette (ABC) transporters ABCC6 and ABCC7. The double mutant of abcc6abcc7 was generated by CRISPR-Cas9 gene editing, but there was no apparent change in phenolic transport into vacuoles. It may be that additional redundancy with other ABCC transporters is obscuring detectable phenotypes (Chapter 4). This thesis illustrates a new paradigm for monolignol export across biological membranes by sink-driven diffusion, and the challenges of investigating monolignol transporters and lignin-modification induced dwarfism.

Text

2022-03-22

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/80992

Botany

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/