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A biochemical and molecular study of lignin biosynthesis Dharmawardhana, Dilsiri Palitha

Abstract

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.

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