Changes in the structure and protein binding ability of condensed tannins during decomposition of fresh needles and leaves

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Abstract

Alterations of the chemical structure of condensed tannins (CT) during decomposition of Norway spruce (Picea abies) needles and white willow (Salix alba) leaves were investigated by gel permeation chromatography (GPC), 13C nuclear magnetic resonance (NMR) spectroscopy, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS). The effect of these alterations on their protein binding capacity was determined by radial diffusion assay on an agarose plate. For these studies an incubation experiment was performed with spruce needles and willow leaves. From the fresh foliage and its decomposed materials obtained after 4-days, and 1-, 2-, 4-, and 8-weeks of incubation, CT were extracted and analyzed.

Dynamics of CT alterations during decomposition of the fresh foliage were different for the two plant species, although the amount of extractable CT for both decreased soon after incubation and only slight amounts of CT were extractable after 8 weeks of incubation. The decrease was faster for the willow leaves than for the spruce needles. Solution 13C NMR revealed alterations of CT isolated from both degrading plant materials, but only to a small extent. However, considerable changes in chemical composition and chain length of the CT were detected by MALDI-TOF MS. Changes in the chemical composition of CT are expected to decrease the protein binding capacity. Applying the radial diffusion assay, this assumption was confirmed for spruce CT, but not for willow CT. This may be explained by (1) higher reactivity of prodelphinidin (PD) than procyanidin (PC), the former is contained in spruce CT but not in willow CT and (2) the slower decomposition rate of spruce needles than willow leaves. Thus, CT in spruce needles may have suffered a greater extent of chemical alteration, which formed partially altered CT with less protein binding capacity. Further, since the decomposition rate of spruce needles was slower than that of willow leaves, the partially altered CT remained extractable for a longer incubation period compared with the willow leave's CT.

Introduction

Tannins are polyphenols that occur in plants where they can amount to 20% of the plant dry weight depending on plants and organs (Hedges and Weliky, 1989, Kelsey and Harmon, 1989, Benner et al., 1990, Terrill et al., 1992, Preston, 1999, Parfitt and Newman, 2000, Yu and Dahlgren, 2000, Hernes et al., 2001). The ecological role of tannins is widely recognized, for example in soils, they reduce potential Al toxicity by binding to Al, but also increase phosphate availability by preventing its adsorption onto clay minerals (Northup et al., 1995a). Due to their ability to form complexes with proteins, exoenzymes can be immobilized, which leads to reduction of their activity, and thus, the velocity of nutrient cycling (Northup et al., 1995a, Northup et al., 1995b, Northup et al., 1998, Schimel et al., 1998, Preston, 1999, Hättenschwiler and Vitousek, 2000). Further, by complexation and precipitation of N-containing compounds, tannins are expected to sequester proteins from microbial degradation. As a result, organic nitrogen is prevented from rapid mineralization, leaching, and utilization for building new microbial and plant biomass (Handley, 1961, Benoit et al., 1968, Baldwin et al., 1983, Horner et al., 1988, Kuiters, 1990, Howard and Howard, 1993, Northup et al., 1995a, Northup et al., 1995b, Schimel et al., 1996, Schimel et al., 1998, Handayanto et al., 1997, Mafongoya et al., 1998, Preston, 1999, Bradley et al., 2000).

Tannins are classified into two subgroups, condensed tannins (CT) and hydrolyzable tannins (HT). CT (or proanthocyanidins) are mixtures of polymers of flavan-3-ol units with different degrees of polymerization and mostly hydroxyl substitution. HT consists of gallic acid, its dimers (hexahydroxydiphenic acid) and its derivatives. Compared to HT, CT are more abundant in woody plants (Hättenschwiler and Vitousek, 2000), are more insoluble (Zucker, 1983, Benner et al., 1990, Hernes et al., 2001) and decompose slower (Lewis and Starkey, 1968). Thus, CT are considered to have an important impact for nitrogen immobilization in soils, in particular during fall when fresh litter materials are introduced into the soil. The efficiency of this immobilization, however, was indicated to be influenced by the chemical composition of CT (Zucker, 1983, Howard and Howard, 1993, Hagerman et al., 1998) and by the chain length of the polymer (Zucker, 1983) as well as by the chemical characteristics of the respective protein (Hagerman and Butler, 1981).

However, little is known about the chemical changes of CT that are occurring during the degradation of fresh plant materials and even less is known about the effect of structural alterations of CT on their protein binding capability. As mentioned above, CT consist of a mixture of different polymers of flavan-3-ol units with different degrees of polymerization and mostly hydroxylation, and their composition differs among plant sources. Considering that these chemical differences may determine their reactivity, it is important to improve our knowledge about this relationship to obtain a better understanding of their ecological role. Due to the diversity of CT from different origin, partially altered CT are also expected to differ in reactivity.

Thus, the objective of this study was to investigate the changes in the chemical structure of CT during the decomposition of CT-containing plant foliage and to relate those chemical alterations to the ability of CT to bind proteins. Therefore, in a model experiment, freeze-dried and meshed fresh foliage of two representative trees, namely Norway spruce and white willow were incubated at 30 °C and their CT were extracted after defined incubation times. The isolated CT were subjected to gel permeation chromatography (GPC) and 13C nuclear magnetic resonance (NMR) spectroscopy. Those techniques, however, can give us only limited information about the size of tannin chains. To circumvent this problem, matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) was applied, which allows the detection of molecules with high molecular masses (from several hundred to several thousand Daltons). Due to the gentle ionization, fragmentation of the macromolecule under research can be avoided.

Section snippets

Incubation

Norway spruce (Picea abies) needles and white willow (Salix alba) leaves were collected from trees in June and September in 2000, respectively. They were separated from the branches with scissors, freeze-dried, and milled (<0.5 mm). A 40 g of foliage sample was mixed with 300 g quartz sand (that was pre-cleaned by soaking into 0.1N HCl for 24 h, followed by a rinsing with deionized water until pH came close to neutral) in a 1000 ml beaker and inoculated with 1 ml aqueous suspension of fresh gardening

Changes in the amount of extractable CT during incubation

After 1 week, approximately 14% of the Ct of the spruce needle sample was lost due to degradation (Table 1). This value increased to 19% after 8 weeks. The willow leaf samples, however, showed a higher decomposition rate. Here, 18 and 31% of the Ct of the fresh material were lost after 1 week and 8 weeks experiment time. The decomposition rate was significantly higher for willow leaves than that for spruce needles (p<0.01). The Nt concentration did not change considerably, while the C-to-N

Discussion

There are some studies on the change in the chemical structure of CT during decomposition of plant materials (Benner et al., 1990, Schofield et al., 1998, Hernes et al., 2001). In these studies, acid depolymerization in the presence of phloroglucinol, and 13C NMR spectroscopy were used. Acid depolymerization has been very successful to estimate the number average molecular weight, PD and PC contents in terminal and extended units, and in addition, to detect other bimolecular compounds such as

Conclusions

In the present study the combination of GPC, liquid-state 13C NMR spectroscopy, and MALDI-TOF MS was applied for the examination of CT during the decomposition of fresh plant materials. In our study, however, fresh foliage that were freeze-dried and meshed before incubation were used to ascertain well defined starting conditions. Following this approach, one has to bear in mind that chemical composition and tannin content differ between green and senescent foliage (Benner et al., 1990, Hernes

Acknowledgements

We thank Prof. Dr C.M. Preston, Pacific Forestry Center, Canada, for giving us valuable advice and suggestions, along with an anonymous reviewer. We also thank M. Penka, who assisted us by the performance of the incubation experiment and the German Science Foundation for financial support.

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