Coniferin, the glucoside of the monolignol coniferyl alcohol, accumulates to high levels in gymnosperms during spring-cambial reactivation. A cinnamyl alcohol glucoside/P-glucosidase system is thought to play a key role i n lignification by releasing the monolignol aglycones. lnvestigation of such an enzyme system in the xylem of Pinus contorta var latifolia Engelm. revealed two major P-glucosidases. One efficiently hydrolyzed the native substrate, coniferin, and the other was more active against synthetic glucosides. l h e coniferin P-glucosidase was purified to apparent homogeneity using anion exchange, hydrophobic interaction, and size-exclusion chromatography. The apparent native molecular weight was estimated to be 60,000. A dominant 28-kD protein and a minor 24-kD protein were detected in the purified preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. lmmunological evidence from polyclonal antibodies directed against the synthetic N-terminal peptide of the 24-kD protein suggested that the native protein is a dimer of 28-kD subunit size. The N-terminal sequence showed that coniferin P-glucosidase has high homology to known plant P-glucosidases. Coniferin, syringin, and a synthetic coniferin analog were preferred substrates for the coniferin 6-glucosidase. In situ localization using the chromogenic coniferin analog showed the exclusive presence of P-glucosidase activity in the differentiating xylem, similar to peroxidase activity.

The developmental timing of lignin biosynthesis and the nature of the polymer have been studied in Arabidopsis thaliana using biochemical and histological techniques. Embryos and developing seedlings were stained with basic fuchsin, and the internal vascular strands were imaged by the optical serial sectioning capability of confocal laser scanning microscopy. Lignin could not be detected in seedlings until approximately 36 h after germination. As the radicle, hypocotyl, and cotyledon expanded, lignification proceeded as a temporally coordinated event closely linked to appearance of spiral or annular thickenings in the developing vasculature. Although A. thaliana does not undergo secondary growth, the plants were extensively lignified by the bolting stage. Chemical analysis demonstrated that Arabidopsis lignin contains primarily guaiacyl units, accompanied by lesser amounts of syringyl structures. Key words: Arabidopsis, lignin, development, confocal microscopy.

Coniferin beta-glucosidase (CBG) catalyzes the hydrolysis of monolignol glucosides to release the cinnamyl alcohols for oxidative polymerization to lignin. Utilizing the N-terminal amino acid sequence of the purified enzyme, the corresponding full-length cDNA sequence was isolated from a Pinus contorta xylem-specific library. The isolated 1909 nucleotide cDNA was confirmed to be that of CBG on the basis of its high homology to family 1 glycosyl hydrolases, the sequence identity with the N-terminal amino acid residues of the purified enzyme, and the coniferin hydrolytic activity and substrate specificity profile displayed by the recombinant protein when expressed in Escherichia coli. The presence of a 23 amino acid N-terminal signal peptide in the deduced 513 amino acid enzyme suggests that CBG is a secretory protein targeted to the ER. The isolation of CBG cDNA will facilitate the evaluation of the importance of this enzyme in the ultimate stages of lignin biosynthesis and could be a valuable tool in manipulating lignin levels in xylem cell walls.

No abstract

No abstract