A [beta]-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/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.

Identification of Glucosyltransferase Genes Involved in Sinapate Metabolism and Lignin Synthesis in Arabidopsis

“…Typically, the glucosides are considered to represent the transport forms of the monolignols, with their respective UGTs and glucosidases acting sequentially in the assembly process. In conifers, the existence of coniferin (coniferyl alcohol-4-O-glucoside) is well established, and recently the gene encoding a coniferin-specific glucosidase has been identified and shown to release the aglycone in vitro (16). To date, however, no gene encoding UGTs of monolignols has been identified from any plant species, and the biochemical work that has been undertaken for more than 20 years has involved partially purified protein fractions (31).…”

“…Sinapyl alcohol-4-O-glucoside, also known as syringin, is considered to be involved in lignin synthesis, since it is thought that glucosylation of the three monolignols (sinapyl alcohol, coniferyl alcohol, and p-coumaryl alcohol) may aid transport of the monomers out of the cell for polymerization into lignin in muro (3). Recently, a specific glucosidase of coniferin (coniferyl alcohol-4-O-glucoside) has been localized at the differentiating xylem, providing some support for these events in lignin assembly (16).…”

Lignin and Lignan Biosynthesis: Distinctions and Reconciliations

“…Indeed, it is puzzling that monolignol glucosides are apparently only found in very few plant species. Thus, the question has not yet been resolved as to whether the monolignol glucosides are required for transportation into lignifying plant cell walls or not, but ongoing work in the Ellis and Savidge laboratories is beginning to bring a substantial measure of clarification to this important subject (83,84).…”

Small Glycosylated Lignin Oligomers Are Stored in Arabidopsis Leaf Vacuoles

“…Since sinapoylglucose:malate sinapoyltransferase, catalyzing the conversion of sinapoyl glucose to sinapoyl malate, is present in the vacuole (Sharma and Strack, 1985;Hause et al, 2002), and because monolignol glucosides can be transported through the tonoplast via ATP binding cassette-like transporters (Miao and Liu, 2010), sinapoyl glucose and the monolignol glucosides are likely stored in the vacuole. A vacuolar localization of coniferin and other monolignol glucosides has been speculated (Leinhos and Savidge, 1993;Dharmawardhana et al, 1995) but has not yet been unambiguously proven (Kaneda et al, 2008).…”