AND LACCASES

None of these three enzyme classes have any demonstrable role in lignification. The early suggestions that a polyphenol oxidase might be involved emerged from studies by Freuden — berg in 1953, using a press—sap extract of the mushroom, Agaricus campestris (272—277) and also later by Mason and Cronyn (278); mushrooms do not, however, biosynthesize lignins. Another candidate was coniferyl alcohol oxidase, detected in jack pine (Pinus strobus) (268, 269), other Pinaceae species (268, 269, 271) and tobacco (270); this has also not been demonstrated to either afford lignins in vitro and/or have a role in lignification in vivo.

As of 2007, there was still no convincing evidence for any direct involvement of laccase in lignification — in spite of numerous articles (12, 53, 54, 258—266) appearing over a five

decade plus time-frame supporting their involvement in lignin macromolecular assembly. None of these studies, however, met the criteria for monolignol oxidation/lignification as set out by Lewis et al. (31), namely, that: “the enzyme must be able to convert mono-, oligo — and polylignols into their free-radical derivatives; the enzyme must be both tem­porally and spatially correlated with sites of lignin biosynthesis; the enzyme, in the pres­ence of the requisite co-factors and/or other proteins, must be demonstrably capable of converting the monolignols into macromolecular lignin chains; and the enzyme must unequivocally be demonstrated as essential for lignin biosynthesis, e. g., through loss of function.”

The evidence for a role of laccases in lignification was scant indeed: originally proposed by Russian workers in the 1940s (258, 259), this was later investigated further in the labora­tories of Freudenberg (12, 53, 54) and Higuchi (260). While laccases are generally capable of oxidizing monolignols, the experiments were not designed at that time to establish if native lignins were being formed with these catalysts. Later, Harkin and Obst (255) reported the exclusive participation of peroxidases in lignification of trees, using the reagent syringal — dazine (66, Figure 7.16). They also concluded that laccases, for example, were absent in Acer (sycamore) species examined based on a lack of histochemical staining. Laccases were, however, subsequently purified from Acerpseudoplatanus (261,263, 267), and the encoding genes cloned (265), thereby disproving their reported absence. On the other hand, as far as putative lignification was concerned, incubation of monolignols with the Acer laccase(s) (263, 267) in vitro only gave preparations with very minor amounts of 8-O-4r interunit linkages that were presumably not polymeric; that is, such preparations did not reflect lignin structure which predominates in 8- O-4r interunit linkages.

An additional study (264), purportedly detecting a laccase in loblolly pine suggested that laccases were also associated with lignification on the basis of staining with syringaldazine (66) and diaminofluorine (67). Furthermore, reaction rates (as measured by rates of oxygen consumption) for p-coumaryl (1), coniferyl (3), and sinapyl (5) alcohols were 5, 72, and 47 nkat mg-1 protein, respectively, with Km values for each either being unobtainable as for p-coumaryl alcohol (1), or very high 12 and 25 mM for coniferyl (3), and sinapyl (5) alcohols, respectively. Such data prompted Ros Barcelo (280) to comment “With these high Km values, it is difficult to imagine what concentration of cinnamyl alcohols it would be necessary to reach its lignifying cell walls to saturate laccase during the oxidation of cinnamyl alcohols to lignin-like compounds.” These data do not therefore represent proof of laccase involvement in lignification.

Later studies (281), attempting to downregulate the laccase multigene family in poplar (Populus trichocarpa), had essentially no effect on lignin contents as estimated by both acetyl bromide and Klason methods (20-25% of CWR), or on lignin compositions as de­termined by thioacidolysis. These findings again indicated that laccases have no significant and/or direct role in monolignol 1/3/5 oxidation leading to macromolecular lignin assem — bly/configuration, in contrast to more than five decades of scientific contributions (12, 53, 54, 258-266) proposing the contrary view.

Interestingly, Arabidopsis has 17 genes encoding laccases, and each of these has been ex­amined for patterns of gene expression using the GUS-reporter system as before (Turlapati et al., manuscript in finalization): eight of these are expressed in vascular (lignifying) tissue(s), although their physiological roles still need to be defined. One of the laccases apparently has a role in seed coat development, with this presumed to be required for condensed tannin formation (282). Yet, at the time of writing, other researchers still continue to suggest that laccases have a role in seed coat lignification (266), even though seed coat tissue apparently does not form lignin.