Current understanding of the final oxidative steps leading to lignin deposition in trees and other higher plants is limited with respect to what enzymes are involved, where they are localized, how they are transported, and what factors regulate them. With the use of cell suspension cultures of sycamore maple (Acer pseudoplatanus), an in-depth study of laccase, one of the oxidative enzymes possibly responsible for catalyzing the dehydrogenative polymerization of monolignols in the extracellular matrix, was undertaken. The time course for secretion of laccase into suspension culture medium was determined with respect to age and mass of the cells. Laccase was completely separated from peroxidase activity by hydrophobic interaction column chromatography, and its purity was assessed with different types of gel electrophoresis (isoelectric focusing-, native-, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Amino acid and glycosyl analyses of the purified enzyme were compared with those reported from previous studies of plant and fungal laccases. The specific activity of laccase toward several common substrates, including monolignols, was determined. Unlike a laccase purified from the Japanese lacquer tree (Rhus vernicifera), laccase from sycamore maple oxidized sinapyl, coniferyl, and p-coumaryl alcohols to form waterinsoluble polymers (dehydrogenation polymers). cambium extracts stimulated DHP formation from coniferyl alcohol more effectively than the addition of H202, from which he concluded that the combined actions of both laccase and peroxidase are important for lignin biosynthesis in plants (12). Subsequent studies of a purified laccase from the Japanese lacquer tree (Rhus vernicifera) found that the enzyme could not oxidize coniferyl alcohol to produce a DHP (26), thus supporting Higuchi's proposal and leading many researchers to believe that laccase plays no role in lignin biosynthesis. Further evidence to support the contention that laccase is not involved in lignin biosynthesis was provided by histochemical studies in which a chromogenic substrate for fungal laccases and peroxidases, syringaldazine, was oxidized when applied to lignifying plant tissues in the presence but not the absence of H202 (15).On the other hand, recent studies of plant laccase and peroxidase activities suggest that syringaldazine oxidation may not be an adequate marker for lignin-specific enzyme activity. In contrast to the results of Harkin and Obst (15), Goldberg et al. (14) found low levels of syringaldazine oxidation in poplar stem tissue sections even without the addition of H202; however, these authors attributed this to low levels of endogenous H202 in the tissues. Additionally, although syringaldazine has been found to be a good substrate for most, if not all, fungal laccases and peroxidases, it is not a substrate for at least one purified plant laccase (18), nor is it a substrate for an extracellular plant peroxidase whose synthesis is correlated with tissue lignification (9). Thus, Nakamura's demonstr...

Abstract.We have investigated the abilities of extracellular enzymes from dark-grown cell-suspension cultures of sycamore maple (Acer pseudoplatanus L.) to oxidize monolignols, the precursors for lignin biosynthesis in plants, as well as a variety of other lignin-related compounds. Laccase and peroxidase both exist as a multiplicity of isoenzymes in filtrates of spent culture medium, but their abilities to produce water-insoluble, dehydrogenation polymers (DHPs) from the monolignols (in the presence of hydrogen peroxide for the peroxidase reaction) appear identical whether or not the enzymes are purified from the concentrated filtrates or left in a crude mixture. The patterns of bonds formed in these DHPs are identical to those found in DHPs synthesized using horseradish peroxidase or fungal laccase, and many of these bonds are found in the natural lignins extracted from different plant sources. On the other hand, sycamore maple laccase is very much less active on phenolic substrates containing multiple aromatic rings than is sycamore maple peroxidase. We suggst that whereas laccase may function during the early stages of lignification to polymerize monolignols into oligo-lignols, cell-wall peroxidases may function when H202 is produced during the later stages of xylem cell development or in response to environmental stresses.

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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