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UBC Theses and Dissertations

Setting the stage for lignin deposition : spatial distribution of enzymes directing lignification in Arabidopsis thaliana Hoffmann, Natalie


Lignin is a critical phenolic polymer that reinforces secondary cell walls of plant cells. Lignin confers structural strength in fiber cells and enables water transportation in xylem vessel elements. Secreted enzymes, laccases (LACs) and peroxidases (PRXs), facilitate lignin polymerization by oxidizing lignin monomers called monolignols. In Arabidopsis thaliana, there are 17 LACs and 73 PRXs and the isoenzymes involved in lignification and their spatial distributions in plant tissues are poorly characterized. This study tested the hypotheses that LACs and PRXs occupy distinctive cell wall domains, and that the activity of these enzymes could enable deposition of lignin to different regions during development. Putative lignin-associated LACs and PRXs, AtLAC4, AtLAC10, AtLAC17, AtPRX42, AtPRX52, AtPRX71, and AtPRX72, were identified using publicly-available mutant and bioinformatics data. Enzymes were fluorescently tagged with an mCherry label and co-localized with lignin, visible as UV autofluorescence. AtLAC4, AtLAC17, and AtPRX72 localized to xylan-rich secondary cell walls of xylem vessel elements and fibers while AtLAC4, AtPRX64, AtPRX71, and AtLAC10 localized to the pectin-rich middle lamella in fibers. AtLAC10, AtPRX42, AtPRX52, and AtPRX71 localized to non-lignified tissues. Enzyme localization may be due to a combination of mobility in the cell wall and timing of expression. Expressing fluorescently-tagged AtLAC4, AtLAC17, and AtPRX64 under the promoter of the secondary cell wall-specific gene AtCESA7 resulted in similar cell wall localization patterns for all three enzymes in roots, indicating that changing the timing of expression was sufficient to alter localization. However, transgenes were transcriptionally silenced in stem tissue. Stem histochemistry demonstrated that both LAC and PRX oxidative activity was restricted to lignifying cells throughout development. Production of apoplastic reactive oxygen species, such as H₂O₂ for PRX activity, was also found exclusively in lignifying tissue and differed temporally between lignified cell types. Taken together, these results indicate that characteristic sets of oxidative enzymes localize to specific cell types and regions of the lignified cell wall. The location of oxidative enzymes and production of oxidative substrates, such as H₂O₂, could ultimately dictate lignin deposition spatially and temporally throughout stem growth.

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