A β-glucosidase from lodgepole pine xylem specific for the lignin precursor coniferin

Abstract

Coniferin, the glucoside of the monolignol coniferyl alcohol, accumulates to high levels in gymnosperms during spring-cambial reactivation. A cinnamyl alcohol glucoside/β-glucosidase system is thought to play a key role in lignification by releasing the monolignol aglycones. Investigation of such an enzyme system in the xylem of Pinus contorta var latifolia Engelm. revealed two major β-glucosidases. One efficiently hydrolyzed the native substrate, coniferin, and the other was more active against synthetic glucosides. The coniferin β-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. Immunological 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 β-glucosidase has high homology to known plant β-glucosidases. Coniferin, syringin, and a synthetic coniferin analog were preferred substrates for the coniferin β-glucosidase. In situ localization using the chromogenic coniferin analog showed the exclusive presence of β-glucosidase activity in the differentiating xylem, similar to peroxidase activity.