The objective of the first half of this thesis was to develop an efficient and
sensitive method to detect lignin in intact tissues using confocal laser scanning
microscopy and to characterize the timing and quality of lignin deposited during
Arabidopsis thaliana development. Lignin could first be detected 48h post-imbibition.
During subsequent development, lignification of vasculature proceeded in a temporally
coordinated manner, with lignin deposition closely following secondary wall thickening
in vascular elements, in a cell-by-cell sequence. A spatially and temporally distinct
pattern of syringyl lignin deposition was observed along the developing stem. Syringyl
type lignin appeared only once fiber differentiation was initiated at day 21.
The second half of the thesis is dedicated to identification, purification and
characterization of a coniferin specific P-glucosidase from Pinus contorta xylem, and
cloning of the corresponding cDNA. Coniferin accumulates to high levels during
cambial reactivation, and a cinnamyl alcohol glucoside/ p-glucosidase system is thought
to play a key role in lignification in gymnosperm tissues. However, evidence for the
existence of P-glucosidases specific for cinnamyl alcohol glucosides in lignifying xylem
has not been conclusive. Using a range of synthetic aromatic P-glucosides and coniferin
as substrates, two major P-glucosidases present in total extractable xylem protein were
identified. The enzyme that demonstrated a high specificity/ activity for coniferin was
purified to homogeneity using anion exchange, hydrophobic interaction and gel
filtration chromatography. Based on the N-terminal amino acid sequence of this
protein, the corresponding cDNA was isolated from a library constructed from
differentiating pine xylem.
A 1909 bp full length cDNA sequence was confirmed to be that of coniferin Pglucosidase
on the basis of it's homology to other plant P-glucosidases and by the
demonstration of coniferin hydrolytic activity and similar substrate specificity when
expressed in E. coli The deduced 513 amino acid protein contains a 23 amino acid N -
terminal signal peptide that is characteristic of eukaryotic secretory proteins. The
isolation of the coniferin |3-glucosidase cDNA offers novel opportunities to clarify the
ultimate steps of lignin biosynthesis. It also opens a route to the possible modification
of lignin in trees by down-regulation of its activity through genetic engineering. / Science, Faculty of / Botany, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/6278 |
| Date | 11 1900 |
| Creators | Dharmawardhana, Dilsiri Palitha |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Format | 12274261 bytes, application/pdf |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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