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Laccase and peroxidase isoenzymes during leaf litter decomposition of Quercus ilex in a Mediterranean ecosystem
Soil Biology & Biochemistry 36 (2004) 1539–1544 www.elsevier.com/locate/soilbio
Laccase and peroxidase isoenzymes during leaf litter decomposition of Quercus ilex in a Mediterranean ecosystem C. Di Nardo, A. Cinquegrana, S. Papa, A. Fuggi, A. Fioretto* Dipartimento di Scienze della Vita, Seconda Universita` di Napoli, Via Vivaldi 43, Caserta 81100, Italy Received in revised form 10 March 2004
Abstract The dynamics of leaf litter decomposition of Quercus ilex (L.) were investigated over a 2 year period by determining the activities and isoenzyme distribution of laccases and peroxidases. The analysis of isoenzymes was performed by isoelectric focusing on high stability pH gradients with high resolving power. The preparation of zymograms was carried out using the leaf litter extract without previous concentration. During litter decomposition, laccase and peroxidase activities changed as well as the type and number of enzyme isoforms. The activities of both enzymes were low (%0.017 and %0.031 mmol o-tolidine oxidized hK1 gK1 d.w. for laccase and peroxidase, respectively) in first year and increased in October–January of the second year of litter decay. The highest activities measured after 15–18 months of litter exposure (0.37G0.03 and 0.19G0.02 mmol o-tolidine oxidized hK1 gK1 d.w. for laccase and peroxidase, respectively), showed that litter chemical composition affected the growth of ligninolytic microbial community. The activation energy for laccase and peroxidase reactions also changed during decomposition: the highest values (55G6 kJ molK1 for laccase and 60G6 kJ molK1 for peroxidase) occurred in autumn–winter, even if spatial changes were evidenced. Some enzyme isoforms (pIZ5.3 and 5.5 for laccase and pIZ5.0 and 5.1 for peroxidase, respectively), contributed more than others to the overall laccase and peroxidase activity, suggesting that some ligninolytic species bloomed in particular seasons of the year, even if other species with similar functional activities colonized the litter. q 2004 Elsevier Ltd. All rights reserved. Keywords: Litter decomposition; Enzyme activity; Laccase; Peroxidase; Isoelectrofocusing
1. Introduction Decomposition of plant litter by the soil microbial community is an important process of controlling nutrient cycling and soil humus formation. Bacteria and fungi decay litter by producing intra- and extracellular enzymes that, therefore, have been assayed for comparing microbial communities and monitoring community succession (Burns, 1978; Sinsabaugh et al., 2002; Heal et al., 1997). The enzymes determining plant litter decomposition rate are mainly the exoenzymes involved in the degradation of ligno-cellulose, that is the major component of plant cell wall (70–80% of litter organic material). Ligno-cellulose also is the main potential energy and nutrient source available to decomposers (Swift et al., 1979). Lignin, in * Corresponding author. Tel.: C39-823-274550; fax: C39-823-274571. E-mail address: [email protected] (A. Fioretto). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.07.013
particular, is a complex organo-aromatic polymer of phenylpropane residues linked by a variety of chemical bonds. This makes it highly refractory and its decay often needs other non-lignified carbon and energy sources (Kirk and Farrell, 1987). Therefore, lignin is less readily available to the decomposer than cellulose and slows down the overall rate of litter decomposition (Berg et al., 1982; Takeda et al., 1987). In addition, lignin decomposition products can form nitrogenous compounds that are very resistant to microbial attack (Berg, 1988). In aerobic environments the most rapid and extensive lignin degradation is caused by fungi, particularly the white-rot fungi (Kirk and Farrell, 1987). Lignin is degraded through oxidative processes that involve peroxidases, polyphenol oxidases and laccases (Gianfreda and Bollag, 2002). These enzymes catalyse the oxidation of lignin, producing phenolic intermediates that can be further degraded to serve as carbon and energy source for microbial
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growth or lead to the formation of complex polymeric humic substances. There are few studies on the microorganisms involved in the production and distribution of phenoloxidases, laccases and peroxidases during decomposition (Sinsabaugh et al., 2002), particularly in the Mediterranean ecosystem (Criquet et al., 1999, 2000). The aim of this study was to monitor laccase and peroxidase enzymes during leaf litter decomposition of the holm-oak (Quercus ilex L.), an evergreen oak of the Mediterranean maquis. It is known that the decomposer community can involve different microorganisms with similar functional activity but expressing different isoenzymes (Sinsabaugh et al., 2002). Isoelectric focusing on pH gradient gel strips has been used to characterize laccase and peroxidase isoenzyme patterns to provide insight on changes in the microbial community contributing to lignin degradation (Osono and Hiroshi, 2001). We also report the temperature response and the activation energy for such enzymes during decomposition.
2. Materials and methods 2.1. Site and soil The experimental plot (3000 m2) is located 200 m a.s.l. with an Eastern exposure in the WWF Oasi Bosco San Silvestro near the city of Caserta (Campania, Italy). The climate in the reserve is typically Mediterranean. There are mild, wet winters and hot, dry summers. The vegetation is mostly Q. ilex L. The soil is calcareous with 70% sand and 27% clay (Papa et al., 2002). The water holding capacity is about 1.56 g H2O gK1 d.w. The pH is 7.0G0.5, and the potential pH (obtained by shaking 10 g of dry soil in 25 ml 1 M KCl) is 6.4. The average content of N, Corg and C/N ratio of soil are 0.50G0.05%, 13.5G1.2% and 26, respectively. In the study plot the slope is interrupted by terracing. 2.2. Sample preparation and processing Freshly abscised leaves of Q. ilex were collected in May– June when most of the litter fall occurs. About 5 g of leaf material, air dried and cleaned by contaminating debris, were placed in each terylene net bag (22!10 cm2 length! height) with a mesh size of 1 mm2. The litter bags (216) were set out in nine randomly selected sites under Q. ilex trees in July 2001. All the bags were fixed on top of the litter layer by metal pegs. Every 3 months, two bags were collected from each site. The litter from each bag was cleaned to remove soil using a small brush and weighted. A subsample of litter (25% fresh weight) from each bag was oven dried at 75 8C and used for dry matter determinations. The remaining litter in the two bags was pooled and used for enzyme extraction.
Due to the high changes in the measured laccase and peroxidase activities, evidencing spatial heterogeneity, the plot was divided into three subplots (three terraces) each including three sampling sites. 2.3. Laccases and peroxidases preparation and assays Laccases (E.C. 1.10.3.2) and peroxidases (E.C. 1.11.1.7) were prepared and assayed according to Leatham and Stahmann (1981) with minor modification: 1 g fine fragmented leaf litter was transferred into a centrifuge tube, suspended in 5 ml 0.05 M acetate pH 5.0 cold buffer and kept at 4 8C. The mixture was homogenised using a Polytron homogeniser for 20–30 s. It was then centrifuged at 27,000g for 10 min at 4 8C. The supernatant fraction was recovered and used as the enzyme extract. The recovery of enzyme activities in the first extraction was 91% of that extractable by three subsequent suspensions of the pellet in the extraction buffer. Therefore only one extraction step was used. Laccase activity in the litter extract was measured by recording the increase of adsorbance at 600 nm for 1 min interval. The reaction mixture contained: 0.1 ml enzyme extract, 0.8 ml 0.05 M pH 5.0 acetate buffer and 0.1 ml 25 mM o-tolidine (3-3 0 dimethyl 4-4 0 diamino biphenyl) in 1 ml of final volume. The assays were performed in a Cary 14 spectrophotometer (Varian, Palo Alto, USA) equipped with Peltier controlled thermocuvettes. Peroxidase activity was determined as the absorbance increased rate when 0.05 ml of 4 mM H2O2 was added in the reaction mixture of laccase. Manganese peroxidase was determined as additional absorbance increase rate when 0.01 ml of 1 M MnSO4 was added to the above reported peroxidase assay mixture. The enzyme assays were performed in triplicate for each litter sample and the activities were expressed as mmol of tolidine oxidized hK1 gK1 of litter d.w., using 6340 as the molar extinction coefficient at 600 nm of tolidine (McClaugherty and Linkins, 1990). 2.4. Arrhenius plot The activation energy for laccase and peroxidase reaction was determined by measuring the enzyme activities at different temperatures and transforming them according to the Arrhenius equation: lnðvÞ Z A K Ea =R !1=T where v is reaction rate; T is the absolute temperature (8K), Ea is the activation energy and R is gas constant. 2.5. Isoelectrofocusing and enzyme assay on strip Non-denaturing isoelectrofocusing (IEF) was performed as previously reported (Sanchez et al., 1997;
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Gallardo et al., 2001), using immobilized pH gradient dry strips (IPG) (Amersham Pharmacia Biotech) but with minor modification. Strips of pH range 3–10 linear and 18 mm long were used. A mixture (0.350 ml) of enzyme crude extract, 2% IPG buffer (carrier ampholyte mixture) and 0.1% bromophenol blue was prepared to rehydrate the dry strips. After 12 h the IPG strip was rinsed with deionized water to remove the excess rehydratation solution and immediately transferred to horizontal flatbed electrophoresis units (Multiphor II system Pharmacia Biotech, Sweden), covered with a paraffin fluid, and submitted to isoelectrofocusing as follows: 18 28 38 48
phase phase phase phase
200 V, 2 mA, 5 W, 25 V/h 500 V, 2 mA, 5 W, 25 V/h 3500 V, 2 mA, 5 W, 3000 V/h 3500 V, 2 mA, 5 W, 30000 V/h
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3.2. Laccase and peroxidase activity during decomposition Laccase and peroxidase activities showed significant spatial differences (P!0.01) among the three terraces of the study site (Fig. 1). Laccase activity was low (%0.017 mmol o-tolidine oxidized hK1 gK1 d.w.) in the first year of decomposition. A significant increase occurred in October after 15 months of decomposition (maximum activity was 0.37G0.03 mmol o-tolidine oxidized hK1 gK1 d.w.) but subsequently decreased and in July, 24 months after exposure, it was !5% of the maximum activity measured in October. Laccase activity showed a similar pattern for subplots 1–3 although maximal activity was significantly higher in subplot 3 (Fig. 1). In January the lowest activity was in subplot 2. Iron peroxidase activity was low in the first year of decomposition but significantly increased from October to
After the electrophoretic course (12 h), the strips were washed rapidly in distilled water to clean them from the cover fluid and submerged (15 ml) in the same reaction mixture used for the laccase and peroxidase enzyme assay. The isoelectric points of laccase and peroxidase were determined in comparison with standard proteins of known isoelectric point processed in the same conditions as the unknown samples.
2.6. Statistics The mass loss over time was fitted to a simple exponential curve (Olson, 1963): lnðxt =xo Þ Z Kkt where xo is the original mass of leaf litter, xt is the amount of litter remaining after time t, t is the time (y) and k is the decomposition rate (yK1). The half decomposition time was also calculated (t1/2Z0.691/k). The significance of differences among the litters was tested by the one-way analysis of variance (ANOVA) followed by the Tukey test. Correlations were determined using the simple Pearson correlation coefficient.
3. Results 3.1. Litter decomposition After 12 months the residual mass was 72G5% of the initial value and, after 24 months, 50G8% (data not presented). The pattern was approximated to an exponential decay function along the overall study period (R2O0.97). The average decay constants over the study period was 0.26G0.03 yK1, corresponding to t1/2 of 2.3 y.
Fig. 1. Laccase (L), iron peroxidase (P), iron and manganese peroxidase (PCMn) activities during Quercus ilex leaf litter decomposition in the three subplots of the experimental stand. Values are meanGSE of three measurements with three replicates. Data outside the scale are annotated with the actual value of the measured activity.
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Fig. 2. Activation energy for laccase (A) and iron peroxidase (B) reactions during Quercus ilex leaf litter decomposition in the three subplots of the experimental stand. Values are meanGSE of three measurements with three replicates.
January (15–18 months). The highest activity occurred in January in subplots 1 and 3, which had the highest laccase activity in October. The highest iron peroxidase activity in subplot 2 occurred in October (0.19G0.02 mmol o-tolidine oxidized hK1 gK1 d.w.) when the compared laccase activity was relatively low. Subsequently activity decreased to its lowest level in the summer (24 months). The contribution of manganese peroxidase to the overall peroxidase activity was always relatively low, but was significant in samples from subplot 3 at 6, 9 and 15 months of decomposition. At 6 months, in particular, the highest activity (0.05G0.01 mmol o-tolidine oxidized hK1 gK1 d.w.) was equal to that of iron peroxidase (Fig. 1). 3.3. Arrhenius plot and activation energy of laccases and peroxidases during litter decomposition Laccase and peroxidase activity were determined in the laboratory at 10, 15, 20, 25 and 30 8C: the range of temperatures monitored in the study site under natural conditions. The Arrhenius plots for the crude enzyme preparation of laccase and peroxidase were linear over the range of temperatures (R2O0.95). The activation energy (Ea) for laccase and peroxidase activity using the crude enzyme preparation changed during decomposition (Fig. 2A and B). The highest Ea for laccases (55G6 kJ molK1) occurred in the enzyme preparations from the October (15 months) samplings when the highest enzyme activity was also measured. The value did not significantly differ between the three subplots. The lowest Ea values (20G2 kJ molK1) occurred in July samplings (12 months)
Fig. 3. Laccase isoforms during Quercus ilex leaf litter decomposition in the three subplots of the experimental stand. Maximal isoform activity during decomposition are reported as bold bands.
when the laccase activity was low (Fig. 1). In this case the subplots showed significant differences (Fig. 3A). The activation energy (Ea) for the peroxidase reaction (Fig. 2B) had the highest values (60G6 kJ molK1) in January samples (18 months) for subplots 1 and 3. At the same time a lower value (30G3 kJ molK1) occurred for subplot 2 which showed the highest peroxidase activity in October (15 months). In these samples the Ea was similar to that found in January in subplot 2. Low activation energy values (15G2 kJ molK1) occurred in April and July when peroxidase activity was low in all the subplots. According the Ea values, the Q10 ranged between 1.2 and 2.2 and 1.2 and 2.6 for laccase and iron peroxidase, respectively. 3.4. Enzyme isoelectrofocusing and assays Laccase and peroxidase activity was tested on the IEF strips after isoelectrofocusing of the crude enzyme extract. Two isoforms for laccase (pI 5.3 and 5.5) were always present in the October and January samplings in all subplots (Fig. 3). In addition, in October (15 months) when the highest activity was detected, there were six isoforms. However, the highest activity was always associated with the 5.3 and 5.5 pI isoforms. The April and July samplings, in contrast,
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Fig. 4. Iron peroxidase isoforms during Quercus ilex leaf litter decomposition in the three subplots of the experimental stand. Maximal isoform activity during decomposition are reported as bold bands.
showed one band at pI 4.9, and 5.1 and 5.3 in subplot 1, 2, 3, respectively, after 9 and 12 months of exposure. The April samplings (21 months) showed an additional band at 4.8. Iron peroxidase showed 2 isoforms at pI 5.0 and 5.1 in the October samplings in all the subplots in both the first and second year of exposure (Fig. 4). Only a single band at pI 5.0 was seen at the other sampling times, and even then in the April and July extracts was very weak. Similar patterns were seen in the three subplots.
4. Discussion Laccases, as well as iron and manganese peroxidases, have been reported to operate during leaf litter decomposition (Kirk and Farrell, 1987; Cameron et al., 2000; Criquet et al., 2000). Apart from manganese peroxidase, that was not always detectable during the decomposition of Q. ilex leaf litter, laccase and iron peroxidase activities were detected all year round (Fig. 1). They were dependent on the stage of decomposition and, probably, on season. The highest activities for laccase and iron peroxidase occurred after 15 and 18 months of litter decomposition, suggesting that the growth of the ligninolytic microflora was restricted
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in the first year, when the more readily degrading litter components favoured the development of more competitive organisms (Berg et al., 1982). This view was supported by the fact that the living fungal biomass colonizing the litter was similar in October through January of the first and second year of decomposition (data not shown). Similar variations for laccase activity during decomposition of Q. ilex litter has been found in Mediterranean ecosystems (Criquet et al., 1999). Peroxidase activity with a maximum in the autumn and winter and a minimum in the summer has been reported for Cistus and Myrtus litter in another Mediterranean maquis area in the same region (Fioretto et al., 2000). A significant increase of laccase activity for such litters also occurred in the autumn. The main laccase and iron peroxidase isoenzymes, detected in our assay conditions, occurred during the wet autumn and winter, while others were present in the dry spring to summer period (Figs. 3 and 4). The lowest enzyme activity in the summer was probably due to the hot and dry condition that would have severely restricted microbial growth (Fioretto et al., 2000). However, apparence of a new laccase isoform (Fig. 1A) in the second year of decomposition suggested that new microorganisms colonized the litter, probably favoured by its variations in physical and chemical composition. Changes in laccase isoforms during decomposition of Q. ilex leaf litter have also been reported in other Mediterranean areas (Criquet et al., 2000). The temperature response of an enzyme reaction will depend on the enzyme structure, as well as its interactions with other low and high molecular substances occurring in the reaction mixture. Differences on the activation energy of an enzyme catalysed reaction, therefore, can suggest differences in enzyme isoforms, as well as in their interactions with compounds present in the reaction environment (Katchalski et al., 1971). Extracellular enzymes in soil, i.e. largely interact with litter colloids and humic compounds, highly affecting their activity and stability (Burns, 1982; Tabatabai and Fu, 1992). The changes in activation energy for the laccase and iron peroxidase reaction did not reflect directly their enzyme isoform distribution (Figs. 2–4), suggesting that other substances synthesized during decomposition could associate with the enzymes and not removed during the extraction procedure and assay. A general increase of Ea related to the increase of soil organic matter humification has been reported (McClaugherty and Linkins, 1990). Soil characteristics and litter status could account of the changes in the isoenzyme patterns (Figs. 3 and 4) as well as in the activation energies (Fig. 2) in the three subplots.
Acknowledgements The research was supported by Second University of Naples. We thank Dr F. Paolella who allowed us to work in the Oasi WWF Bosco di S. Silvestro, Caserta.
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References Berg, B., Hannus, K., Popoff, T., Theander, O., 1982. Changes in organic chemical components of needle litter during decomposition. Longterm decomposition in a Scots pine forest I. Canadian Journal of Botany 60, 1310–1319. Berg, B., 1988. Dynamics of nitrogen (15N) in decomposing Scots pine (Pinus sylvestris) needle litter. Long-term decomposition in a Scots pine forest VI. Canadian Journal of Botany 66, 1539–1546. Burns, R.G., 1978. Enzyme activity in soil: some theoretical and practical considerations, in: Burns, R.G. (Ed.), Soil Enzymes. Academic Press, London, pp. 295–340. Burns, R.G., 1982. Enzyme activities in soil: location and a possible role in microbial ecology. Soil Biology & Biochemistry 14, 423–427. Cameron, M.D., Timofeevski, S., Aust, S.D., 2000. Enzymology of Phanerochaete chrysosporium with respect to the degradation of recalcitrant compounds and xenobiotics. Applied Microbiology and Biotechnology 54, 751–758. Criquet, S., Tagger, S., Vogt, G., Iacazio, G., Le Petit, J., 1999. Laccase activity of forest litter. Soil Biology & Biochemistry 31, 1239–1244. Criquet, S., Farnet, A.M., Tagger, S., Le Petit, J., 2000. Annual variations of phenoloxidase activities in an evergreen oak litter:influence of certain biotic and abiotic factors. Soil Biology & Biochemistry 32, 1505–1513. Fioretto, A., Papa, S., Curcio, E., Sorrentino, G., Fuggi, A., 2000. Enzyme dynamics on decomposing leaf litter of Cistus incanus and Myrtus communis in Mediterranean ecosystem. Soil Biology & Biochemistry 32, 1847–1855. Gallardo, K., Job, C., Groot, P.C.S., Puype, M., Demol, H., Vandekerckhove, J., Job, D., 2001. Proteomic analysis of arabidopsis seed germination and priming. Plant Physiology 126, 835–848. Gianfreda, L., Bollag, J.M., 2002. Isolated enzymes for the transformation and detoxification of organic pollutants, in: Burns, R.G., Dick, R.P. (Eds.), Enzymes in the Environment. Marcel Dekker, New York, pp. 267–284. Heal, O.W., Anderson, J.M., Swift, M.J., 1997. Plant litter quality and decomposition: an historical overview, in: Cadish, G., Giller, K.E. (Eds.), Driven by Nature, Plant Litter Quality and Decomposition. CAB International, Cambridge, pp. 3–30.
Katchalski, E., Silman, I., Goldman, R., 1971. Effect of the microenvironment on the mode of action of immobilized enzymes. Advances in Enzymology 28, 506–511. Kirk, T.K., Farrell, R.L., 1987. Enzymatic combustion: the microbial degradation of lignin. Annual Review of Microbiology 41, 465–505. Leatham, G.F., Stahmann, M.A., 1981. Studies on the laccase of Lentinus edodes: specificy, localization and association with the development of fruiting bodies. Journal of General Microbiology 125, 147–157. McClaugherty, C.A., Linkins, A.E., 1990. Temperature responses of enzymes in two forest soils. Soil Biology & Biochemistry 22, 29–33. Olson, J., 1963. Energy storage and balance of producers and decomposers in ecological systems. Ecology 44, 322–331. Osono, T., Hiroshi, T., 2001. Organic chemical and nutrient dynamics in decomposing beech leaf litter in relation to fungal ingrowth and succession during 3-year decomposition processes in a cool temperate deciduous forest in Japan. Ecological Research 16, 649–670. Papa, S., Curcio, E., Lombardi, A., D’Oriano, P., Fioretto, A., 2002. Soil microbial activity in three evergreen oak (Quercus ilex) woods in a Mediterranean area, in: Violante, A., Huang, P.M., Bollag, J.M., Gianfreda, L. (Eds.), Development in Soil Science 28B. Elsevier Science, Amsterdam, pp. 229–237. Sanchez, J.C., Rouge, V., Pisteur, M., Ravier, F., Tonella, L., Moonsmayer, M., Wilkins, M.R., Hochstrasser, D.F., 1997. Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 18, 324–327. Sinsabaugh, R.L., Carreiro, M.M., Alvarez, S., 2002. Enzyme and microbial dynamics of litter decomposition, in: Burns, R.G., Dick, R.P. (Eds.), Enzymes in the Environment. Marcel Dekker, New York, pp. 249–265. Swift, M.J., Heal, O.W., Anderson, J.M., 1979. Decomposition in Terrestrial Ecosystems. Blackwell Scientific Publications, Oxford/London/Edinburg/Melbourne. Tabatabai, M.A., Fu, M., 1992. Extraction of enzymes from soils, in: Stotzky, G., Bollag, J.-M. (Eds.), Soil Biochemistry, vol. 7. Marcel Dekker, New York, pp. 197–227. Takeda, H., Ishida, Y., Tsutsumi, T., 1987. Decomposition of leaf litter in relation to litter quality and site conditions. Memoirs of the College of Agriculture, Kyoto University 130, 17–38.
Laccase and peroxidase isoenzymes during leaf litter decomposition of Quercus ilex in a Mediterranean ecosystem C. Di Nardo, A. Cinquegrana, S. Papa, A. Fuggi, A. Fioretto* Dipartimento di Scienze della Vita, Seconda Universita` di Napoli, Via Vivaldi 43, Caserta 81100, Italy Received in revised form 10 March 2004
Abstract The dynamics of leaf litter decomposition of Quercus ilex (L.) were investigated over a 2 year period by determining the activities and isoenzyme distribution of laccases and peroxidases. The analysis of isoenzymes was performed by isoelectric focusing on high stability pH gradients with high resolving power. The preparation of zymograms was carried out using the leaf litter extract without previous concentration. During litter decomposition, laccase and peroxidase activities changed as well as the type and number of enzyme isoforms. The activities of both enzymes were low (%0.017 and %0.031 mmol o-tolidine oxidized hK1 gK1 d.w. for laccase and peroxidase, respectively) in first year and increased in October–January of the second year of litter decay. The highest activities measured after 15–18 months of litter exposure (0.37G0.03 and 0.19G0.02 mmol o-tolidine oxidized hK1 gK1 d.w. for laccase and peroxidase, respectively), showed that litter chemical composition affected the growth of ligninolytic microbial community. The activation energy for laccase and peroxidase reactions also changed during decomposition: the highest values (55G6 kJ molK1 for laccase and 60G6 kJ molK1 for peroxidase) occurred in autumn–winter, even if spatial changes were evidenced. Some enzyme isoforms (pIZ5.3 and 5.5 for laccase and pIZ5.0 and 5.1 for peroxidase, respectively), contributed more than others to the overall laccase and peroxidase activity, suggesting that some ligninolytic species bloomed in particular seasons of the year, even if other species with similar functional activities colonized the litter. q 2004 Elsevier Ltd. All rights reserved. Keywords: Litter decomposition; Enzyme activity; Laccase; Peroxidase; Isoelectrofocusing
1. Introduction Decomposition of plant litter by the soil microbial community is an important process of controlling nutrient cycling and soil humus formation. Bacteria and fungi decay litter by producing intra- and extracellular enzymes that, therefore, have been assayed for comparing microbial communities and monitoring community succession (Burns, 1978; Sinsabaugh et al., 2002; Heal et al., 1997). The enzymes determining plant litter decomposition rate are mainly the exoenzymes involved in the degradation of ligno-cellulose, that is the major component of plant cell wall (70–80% of litter organic material). Ligno-cellulose also is the main potential energy and nutrient source available to decomposers (Swift et al., 1979). Lignin, in * Corresponding author. Tel.: C39-823-274550; fax: C39-823-274571. E-mail address: [email protected] (A. Fioretto). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.07.013
particular, is a complex organo-aromatic polymer of phenylpropane residues linked by a variety of chemical bonds. This makes it highly refractory and its decay often needs other non-lignified carbon and energy sources (Kirk and Farrell, 1987). Therefore, lignin is less readily available to the decomposer than cellulose and slows down the overall rate of litter decomposition (Berg et al., 1982; Takeda et al., 1987). In addition, lignin decomposition products can form nitrogenous compounds that are very resistant to microbial attack (Berg, 1988). In aerobic environments the most rapid and extensive lignin degradation is caused by fungi, particularly the white-rot fungi (Kirk and Farrell, 1987). Lignin is degraded through oxidative processes that involve peroxidases, polyphenol oxidases and laccases (Gianfreda and Bollag, 2002). These enzymes catalyse the oxidation of lignin, producing phenolic intermediates that can be further degraded to serve as carbon and energy source for microbial
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growth or lead to the formation of complex polymeric humic substances. There are few studies on the microorganisms involved in the production and distribution of phenoloxidases, laccases and peroxidases during decomposition (Sinsabaugh et al., 2002), particularly in the Mediterranean ecosystem (Criquet et al., 1999, 2000). The aim of this study was to monitor laccase and peroxidase enzymes during leaf litter decomposition of the holm-oak (Quercus ilex L.), an evergreen oak of the Mediterranean maquis. It is known that the decomposer community can involve different microorganisms with similar functional activity but expressing different isoenzymes (Sinsabaugh et al., 2002). Isoelectric focusing on pH gradient gel strips has been used to characterize laccase and peroxidase isoenzyme patterns to provide insight on changes in the microbial community contributing to lignin degradation (Osono and Hiroshi, 2001). We also report the temperature response and the activation energy for such enzymes during decomposition.
2. Materials and methods 2.1. Site and soil The experimental plot (3000 m2) is located 200 m a.s.l. with an Eastern exposure in the WWF Oasi Bosco San Silvestro near the city of Caserta (Campania, Italy). The climate in the reserve is typically Mediterranean. There are mild, wet winters and hot, dry summers. The vegetation is mostly Q. ilex L. The soil is calcareous with 70% sand and 27% clay (Papa et al., 2002). The water holding capacity is about 1.56 g H2O gK1 d.w. The pH is 7.0G0.5, and the potential pH (obtained by shaking 10 g of dry soil in 25 ml 1 M KCl) is 6.4. The average content of N, Corg and C/N ratio of soil are 0.50G0.05%, 13.5G1.2% and 26, respectively. In the study plot the slope is interrupted by terracing. 2.2. Sample preparation and processing Freshly abscised leaves of Q. ilex were collected in May– June when most of the litter fall occurs. About 5 g of leaf material, air dried and cleaned by contaminating debris, were placed in each terylene net bag (22!10 cm2 length! height) with a mesh size of 1 mm2. The litter bags (216) were set out in nine randomly selected sites under Q. ilex trees in July 2001. All the bags were fixed on top of the litter layer by metal pegs. Every 3 months, two bags were collected from each site. The litter from each bag was cleaned to remove soil using a small brush and weighted. A subsample of litter (25% fresh weight) from each bag was oven dried at 75 8C and used for dry matter determinations. The remaining litter in the two bags was pooled and used for enzyme extraction.
Due to the high changes in the measured laccase and peroxidase activities, evidencing spatial heterogeneity, the plot was divided into three subplots (three terraces) each including three sampling sites. 2.3. Laccases and peroxidases preparation and assays Laccases (E.C. 1.10.3.2) and peroxidases (E.C. 1.11.1.7) were prepared and assayed according to Leatham and Stahmann (1981) with minor modification: 1 g fine fragmented leaf litter was transferred into a centrifuge tube, suspended in 5 ml 0.05 M acetate pH 5.0 cold buffer and kept at 4 8C. The mixture was homogenised using a Polytron homogeniser for 20–30 s. It was then centrifuged at 27,000g for 10 min at 4 8C. The supernatant fraction was recovered and used as the enzyme extract. The recovery of enzyme activities in the first extraction was 91% of that extractable by three subsequent suspensions of the pellet in the extraction buffer. Therefore only one extraction step was used. Laccase activity in the litter extract was measured by recording the increase of adsorbance at 600 nm for 1 min interval. The reaction mixture contained: 0.1 ml enzyme extract, 0.8 ml 0.05 M pH 5.0 acetate buffer and 0.1 ml 25 mM o-tolidine (3-3 0 dimethyl 4-4 0 diamino biphenyl) in 1 ml of final volume. The assays were performed in a Cary 14 spectrophotometer (Varian, Palo Alto, USA) equipped with Peltier controlled thermocuvettes. Peroxidase activity was determined as the absorbance increased rate when 0.05 ml of 4 mM H2O2 was added in the reaction mixture of laccase. Manganese peroxidase was determined as additional absorbance increase rate when 0.01 ml of 1 M MnSO4 was added to the above reported peroxidase assay mixture. The enzyme assays were performed in triplicate for each litter sample and the activities were expressed as mmol of tolidine oxidized hK1 gK1 of litter d.w., using 6340 as the molar extinction coefficient at 600 nm of tolidine (McClaugherty and Linkins, 1990). 2.4. Arrhenius plot The activation energy for laccase and peroxidase reaction was determined by measuring the enzyme activities at different temperatures and transforming them according to the Arrhenius equation: lnðvÞ Z A K Ea =R !1=T where v is reaction rate; T is the absolute temperature (8K), Ea is the activation energy and R is gas constant. 2.5. Isoelectrofocusing and enzyme assay on strip Non-denaturing isoelectrofocusing (IEF) was performed as previously reported (Sanchez et al., 1997;
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Gallardo et al., 2001), using immobilized pH gradient dry strips (IPG) (Amersham Pharmacia Biotech) but with minor modification. Strips of pH range 3–10 linear and 18 mm long were used. A mixture (0.350 ml) of enzyme crude extract, 2% IPG buffer (carrier ampholyte mixture) and 0.1% bromophenol blue was prepared to rehydrate the dry strips. After 12 h the IPG strip was rinsed with deionized water to remove the excess rehydratation solution and immediately transferred to horizontal flatbed electrophoresis units (Multiphor II system Pharmacia Biotech, Sweden), covered with a paraffin fluid, and submitted to isoelectrofocusing as follows: 18 28 38 48
phase phase phase phase
200 V, 2 mA, 5 W, 25 V/h 500 V, 2 mA, 5 W, 25 V/h 3500 V, 2 mA, 5 W, 3000 V/h 3500 V, 2 mA, 5 W, 30000 V/h
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3.2. Laccase and peroxidase activity during decomposition Laccase and peroxidase activities showed significant spatial differences (P!0.01) among the three terraces of the study site (Fig. 1). Laccase activity was low (%0.017 mmol o-tolidine oxidized hK1 gK1 d.w.) in the first year of decomposition. A significant increase occurred in October after 15 months of decomposition (maximum activity was 0.37G0.03 mmol o-tolidine oxidized hK1 gK1 d.w.) but subsequently decreased and in July, 24 months after exposure, it was !5% of the maximum activity measured in October. Laccase activity showed a similar pattern for subplots 1–3 although maximal activity was significantly higher in subplot 3 (Fig. 1). In January the lowest activity was in subplot 2. Iron peroxidase activity was low in the first year of decomposition but significantly increased from October to
After the electrophoretic course (12 h), the strips were washed rapidly in distilled water to clean them from the cover fluid and submerged (15 ml) in the same reaction mixture used for the laccase and peroxidase enzyme assay. The isoelectric points of laccase and peroxidase were determined in comparison with standard proteins of known isoelectric point processed in the same conditions as the unknown samples.
2.6. Statistics The mass loss over time was fitted to a simple exponential curve (Olson, 1963): lnðxt =xo Þ Z Kkt where xo is the original mass of leaf litter, xt is the amount of litter remaining after time t, t is the time (y) and k is the decomposition rate (yK1). The half decomposition time was also calculated (t1/2Z0.691/k). The significance of differences among the litters was tested by the one-way analysis of variance (ANOVA) followed by the Tukey test. Correlations were determined using the simple Pearson correlation coefficient.
3. Results 3.1. Litter decomposition After 12 months the residual mass was 72G5% of the initial value and, after 24 months, 50G8% (data not presented). The pattern was approximated to an exponential decay function along the overall study period (R2O0.97). The average decay constants over the study period was 0.26G0.03 yK1, corresponding to t1/2 of 2.3 y.
Fig. 1. Laccase (L), iron peroxidase (P), iron and manganese peroxidase (PCMn) activities during Quercus ilex leaf litter decomposition in the three subplots of the experimental stand. Values are meanGSE of three measurements with three replicates. Data outside the scale are annotated with the actual value of the measured activity.
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Fig. 2. Activation energy for laccase (A) and iron peroxidase (B) reactions during Quercus ilex leaf litter decomposition in the three subplots of the experimental stand. Values are meanGSE of three measurements with three replicates.
January (15–18 months). The highest activity occurred in January in subplots 1 and 3, which had the highest laccase activity in October. The highest iron peroxidase activity in subplot 2 occurred in October (0.19G0.02 mmol o-tolidine oxidized hK1 gK1 d.w.) when the compared laccase activity was relatively low. Subsequently activity decreased to its lowest level in the summer (24 months). The contribution of manganese peroxidase to the overall peroxidase activity was always relatively low, but was significant in samples from subplot 3 at 6, 9 and 15 months of decomposition. At 6 months, in particular, the highest activity (0.05G0.01 mmol o-tolidine oxidized hK1 gK1 d.w.) was equal to that of iron peroxidase (Fig. 1). 3.3. Arrhenius plot and activation energy of laccases and peroxidases during litter decomposition Laccase and peroxidase activity were determined in the laboratory at 10, 15, 20, 25 and 30 8C: the range of temperatures monitored in the study site under natural conditions. The Arrhenius plots for the crude enzyme preparation of laccase and peroxidase were linear over the range of temperatures (R2O0.95). The activation energy (Ea) for laccase and peroxidase activity using the crude enzyme preparation changed during decomposition (Fig. 2A and B). The highest Ea for laccases (55G6 kJ molK1) occurred in the enzyme preparations from the October (15 months) samplings when the highest enzyme activity was also measured. The value did not significantly differ between the three subplots. The lowest Ea values (20G2 kJ molK1) occurred in July samplings (12 months)
Fig. 3. Laccase isoforms during Quercus ilex leaf litter decomposition in the three subplots of the experimental stand. Maximal isoform activity during decomposition are reported as bold bands.
when the laccase activity was low (Fig. 1). In this case the subplots showed significant differences (Fig. 3A). The activation energy (Ea) for the peroxidase reaction (Fig. 2B) had the highest values (60G6 kJ molK1) in January samples (18 months) for subplots 1 and 3. At the same time a lower value (30G3 kJ molK1) occurred for subplot 2 which showed the highest peroxidase activity in October (15 months). In these samples the Ea was similar to that found in January in subplot 2. Low activation energy values (15G2 kJ molK1) occurred in April and July when peroxidase activity was low in all the subplots. According the Ea values, the Q10 ranged between 1.2 and 2.2 and 1.2 and 2.6 for laccase and iron peroxidase, respectively. 3.4. Enzyme isoelectrofocusing and assays Laccase and peroxidase activity was tested on the IEF strips after isoelectrofocusing of the crude enzyme extract. Two isoforms for laccase (pI 5.3 and 5.5) were always present in the October and January samplings in all subplots (Fig. 3). In addition, in October (15 months) when the highest activity was detected, there were six isoforms. However, the highest activity was always associated with the 5.3 and 5.5 pI isoforms. The April and July samplings, in contrast,
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Fig. 4. Iron peroxidase isoforms during Quercus ilex leaf litter decomposition in the three subplots of the experimental stand. Maximal isoform activity during decomposition are reported as bold bands.
showed one band at pI 4.9, and 5.1 and 5.3 in subplot 1, 2, 3, respectively, after 9 and 12 months of exposure. The April samplings (21 months) showed an additional band at 4.8. Iron peroxidase showed 2 isoforms at pI 5.0 and 5.1 in the October samplings in all the subplots in both the first and second year of exposure (Fig. 4). Only a single band at pI 5.0 was seen at the other sampling times, and even then in the April and July extracts was very weak. Similar patterns were seen in the three subplots.
4. Discussion Laccases, as well as iron and manganese peroxidases, have been reported to operate during leaf litter decomposition (Kirk and Farrell, 1987; Cameron et al., 2000; Criquet et al., 2000). Apart from manganese peroxidase, that was not always detectable during the decomposition of Q. ilex leaf litter, laccase and iron peroxidase activities were detected all year round (Fig. 1). They were dependent on the stage of decomposition and, probably, on season. The highest activities for laccase and iron peroxidase occurred after 15 and 18 months of litter decomposition, suggesting that the growth of the ligninolytic microflora was restricted
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in the first year, when the more readily degrading litter components favoured the development of more competitive organisms (Berg et al., 1982). This view was supported by the fact that the living fungal biomass colonizing the litter was similar in October through January of the first and second year of decomposition (data not shown). Similar variations for laccase activity during decomposition of Q. ilex litter has been found in Mediterranean ecosystems (Criquet et al., 1999). Peroxidase activity with a maximum in the autumn and winter and a minimum in the summer has been reported for Cistus and Myrtus litter in another Mediterranean maquis area in the same region (Fioretto et al., 2000). A significant increase of laccase activity for such litters also occurred in the autumn. The main laccase and iron peroxidase isoenzymes, detected in our assay conditions, occurred during the wet autumn and winter, while others were present in the dry spring to summer period (Figs. 3 and 4). The lowest enzyme activity in the summer was probably due to the hot and dry condition that would have severely restricted microbial growth (Fioretto et al., 2000). However, apparence of a new laccase isoform (Fig. 1A) in the second year of decomposition suggested that new microorganisms colonized the litter, probably favoured by its variations in physical and chemical composition. Changes in laccase isoforms during decomposition of Q. ilex leaf litter have also been reported in other Mediterranean areas (Criquet et al., 2000). The temperature response of an enzyme reaction will depend on the enzyme structure, as well as its interactions with other low and high molecular substances occurring in the reaction mixture. Differences on the activation energy of an enzyme catalysed reaction, therefore, can suggest differences in enzyme isoforms, as well as in their interactions with compounds present in the reaction environment (Katchalski et al., 1971). Extracellular enzymes in soil, i.e. largely interact with litter colloids and humic compounds, highly affecting their activity and stability (Burns, 1982; Tabatabai and Fu, 1992). The changes in activation energy for the laccase and iron peroxidase reaction did not reflect directly their enzyme isoform distribution (Figs. 2–4), suggesting that other substances synthesized during decomposition could associate with the enzymes and not removed during the extraction procedure and assay. A general increase of Ea related to the increase of soil organic matter humification has been reported (McClaugherty and Linkins, 1990). Soil characteristics and litter status could account of the changes in the isoenzyme patterns (Figs. 3 and 4) as well as in the activation energies (Fig. 2) in the three subplots.
Acknowledgements The research was supported by Second University of Naples. We thank Dr F. Paolella who allowed us to work in the Oasi WWF Bosco di S. Silvestro, Caserta.
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