Methods for Determining Location

Spot tests, either of liquid culture samples or cultures grown on solid media, have relied on the use of colored products formed during enzymatic oxidations. Some of these tests have been used to distinguish white rot fungi from brown rot fungi and have been used for taxo-nomic classification. Immunocytochemical location of the fungal laccase from Rigidoporous lignosus grown on wood was carried out using antilaccase polyclonal sera and immunogold labeling (17). These studies found laccase to be located in fungal cytoplasm, in vesicle-like structures at the plasmalemma, in the cell wall, and in the extracellular slime layer connecting fungal cells to wood. In liquid-grown cultures of the fungus Coprinus congregatus, laccase located in the hyphal tips was suggested to be involved with sclero-tia/primordia formation (18).

In plants, cytochemical localization of laccase has relied upon the use of syringaldazine, 2,7-diamino-fluorene (DAF), 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate; ABTS), and 4-methylcatechol as substrates. Laccase was localized to active lignifying zones of xylem cells in loblolly pine sections using DAF as a substrate (19). Similarly, cytochemical localization of laccase in tobacco stem cells was found in the outermost lignifying zones in xylem tissue (20). Driouch et al. (21) found laccase excreted in the extracellular medium and associated with cell walls in sycamore cell cultures by cytochemical and immuno-cytochemical methods. In contrast, De Marco and Roubelakis-Angelakis (22) observed laccase in areas where lignification was thought to occur in tobacco protoplasts.

Tissue printing of stem cross sections using 4-methyl catechol and syringaldazine as cytochemical substrates

Figure 1 Types of reactions catalyzed by laccases. Reactions appearing in (a)-(c) were taken from Sanchez-Amat and Solano (16) and reactions appearing in (d)-(f) were taken from Solomon et al. (6).
Figure 2 Chemical structures of substrates used to monitor laccase activity.

coupled with immunolabeling located laccase in the epidermal tissue (21). In petiole tissues, laccase was located in lignifying cell walls of xylem and epidermal cells. All cytochemical methods must employ some method to distinguish peroxidase and tyrosinase (catechol oxidase, phenol oxidase) from laccase. Peroxidase can be distinguished from laccase by samples treated with and without hydrogen peroxide and by including catalase in the incubation medium to remove hydrogen peroxide. A variety of tyrosinase inhibitors (i.e., tropo-lone, SHAM (salicylhydroxamic acid), 4-hexylresorci-nol) can be included in the incubation medium to block tyrosinase activity without affecting laccase activity.

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