Analysis and decomposition of condensed tannins in tree leaves

In the past decades, many studies have shown that tannins (polyphenolic compounds) are involved in defence mechanisms of plants against attack by bacteria, fungi, and herbivores. Little is known about the mechanisms of action of the polyphenolic compounds (Zucker 1983, Scalbert 1991) even though modern analytical methods have improved the analysis of these complex structures (Mole and Watermann 1987, Schofield et al. 2001). Proposals on the mechanism of action include tannins forming stable complexes with plant proteins to make the tissue unattractive and difficult to digest (Schofield et al. 2001), and tannins acting like a toxin through highly specific reactions with digestive enzymes or directly at the cell membranes (Zucker 1983) or through depletion of essential iron by complexation (Mila et al. 1996). Such mechanisms are not only important as a defence in living plants, but may also control terrestrial and aquatic decomposition processes, provided that the effective substances remain in the dead tissues. Indeed, leaves with high initial contents of polyphenolic compounds, especially of condensed tannins (proanthocyanidins), seem to decompose slowly in both terrestrial (Valachovic et al. 2004) and aquatic ecosystems (Wantzen et al. 2002). However, only strong relationships but not significant correlations between the initial content of total polyphenolic compounds or tannins and the velocity of litter decomposition have been demonstrated in most studies, which indicates that multiple causes of delayed decomposition are likely. The analytical methods for the quantitative determination of polyphenolic compounds have been reviewed (Mole and Waterman 1987, Hagermann 1998, Schofield et al. 2001). Simple photometric methods for the determination of total phenols have been preferred over substance-specific methods, e.g., for proanthocyanidins, in ecological studies on leaf litter decomposition. Such general methods, however, have considerable drawbacks (Schofield et al. 2001). Both the isolation and the precise analysis of proanthocyanidins are very complex and timeconsuming. Hence, nearly all of the studies report only photometrically determined initial contents of total phenols. Initial values of proanthocyanidins or other fractions of polyphenols have been reported in some studies, but proanthocyanidin degradation over time has not been analysed. Proanthocyanidins may play an important role in aquatic leaf litter decomposition, as they may deter invertebrate shredders (Wantzen et al. 2002). Moreover, they may retard the "conditioning" of leaves by fungi and bacteria. To test this hypothesis, analytical data of the long-term decomposition of leaf litter, e.g., analysis of the change in the content of proanthocyanidins in decomposing plant material, are needed. Here we describe a method to extract and precisely determine proanthocyanidins from decomposing tree leaves. The method is used to follow the decomposition over time of the leaves of three tree species common to three tropical aquatic environments. The decomposition of leaf litter is controlled by several factors. One factor that may play an important role is the content of condensed tannins (proanthocyanidins). Here we designed a combined method to isolate proanthocyanidins from leaf extracts, to convert them to anthocyanidins, and to quantify individual anthocyanidins exactly with a new simple, but sensitive highperformance liquid chromatography method. We used this method to show composition of proanthocyanidins and to monitor degradation of proanthocyanidins and individual constituents in leaf litter in an aquatic environment over time. Despite the rapid decrease in the initial concentrations, a fraction of the proanthocyanidins remained detectable for several weeks.