Biochemical characterization, molecular cloning and expression of laccases – a divergent gene family – in poplar

Abstract

The nature of the enzyme(s) involved in the dehydrogenative polymerization of lignin monomers is still a matter of debate. Potential candidates include laccases which have recently received attention due to their capacity to oxidize lignin monomers and close spatial and temporal correlation with lignin deposition. We have characterized two H 2 O 2 -independent phenoloxidases with approximate molecular masses of 90 kDa and 110 kDa from cell walls of Populus euramericana xylem that are capable of oxidizing coniferyl alcohol. The 90-kDa protein was purified to apparent homogeneity and extensively characterized at the biochemical and structural levels. To our knowledge, this is the first report of a plant laccase purified to homogeneity from a lignifying tissue of an angiosperm.The cDNA clones corresponding to the 90-kDa and 110-kDa proteins, lac90 and lac110, were obtained by a PCRbased approach using specific oligonucleotides derived from peptide sequences. Sequence analysis indicated that lac90 and lac110 encoded two distinct laccases. In addition, heterologous screening using an Acer pseudoplatanus laccase cDNA enabled us to obtain three additional cDNAs (lac1, lac2, lac3) that did not correspond to lac90 and lac110. The five laccase cDNAs correspond to a highly divergent multigene family but Northern analysis with genespecific probes indicated that all of the genes are exclusively and abundantly expressed in stems. These results highlight the polymorphism of plant laccases by an integrated biochemical and molecular approach, and provide the tools that will enable us to clearly determine the function of these enzymes in plants by molecular and genetic approaches.Keywords: laccase, poplar, lignification, dehydrogenation polymers.Lignins are complex aromatic heteropolymers that are derived from phenylpropanoid metabolism. They represent 30% of the Earth's woody biomass and are considered to have had a crucial role in the adaptation of land plants to the terrestrial habitat. However, many aspects concerning their biosynthesis and its regulation have yet to be elucidated. One striking illustration of this is that the enzyme(s) involved in the final polymerization of the p-hydroxycinnamyl alcohol precursors (monolignols) is still unknown. Freudenberg et al.[1] were first to demonstrate that a laccase (p-diphenol:oxygen oxidoreductase, EC 1.10.3.2) from extracts of the fungus, Agaricus campestris, and a laccase-like enzyme from cambial extracts of the gymnosperm, Araucaria excelsa, could catalyse coniferyl alcohol polymerization, thereby implicating laccases in lignification. However, when Nakamura [2] reported that laccase from Rhus vernicifera could not oxidize coniferyl alcohol and Harkin and Obst [3] showed that tree stems sections could not oxidize syringaldazine in the absence of exogenous H 2 O 2 , the proposed involvement of laccase in lignification was discounted. Peroxidase was thereafter considered to be exclusively involved in the dehydrogenative polymerization of lignin precursors.In recent years, convinc...

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