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Comparison of cellulase activity on decomposing leaves in a hardwood forest and woodland stream
Soi/ Bid. Btochem. Vol. 22. No. 3, pp. 423425. Pnntcd in Great Britain. All rights reserved
0038-0717/90s3.w + 0.00
1990
Copyright c 1990Pcrgamon Press pie
COMPARISON OF CELLULASE ACTIVITY ON DECOMPOSING LEAVES IN A HARDWOOD FOREST AND WOODLAND STREAM A. E. LINKIX and R. L. SINSABAUGH’ Biology apartment,
Clarkson University, Fotsdam, NY 13676, U.S.A.
Biology Department,
Mount Union College, Alliance, OH 44601, U.S.A.
C. A. MCCLAUGHERTY
J. M. MELILLO Tfre Ecosystem Center, Marine Biological Laboratory, Woods Hofe, MA 02543, U.S.A. (Accepted 15 August 1989)
Decomposing leaf litter plays a key role in nutrient cycling and soil organic matter dynamics in forested ecosystems. The initial decomposition of plant fiber is an extracellular process principally mediated by a spectrum of extraceflular enzymes (Burns, 1983). The activity of these enzymes is inffuenced by environmental variables including temperature, moisture and pH as well as the physical and chemical nature of the enzymes and substrates involved (Sinsabaugh er ul., 1981; Gressel et al., l983; Linkins et al., 1984; Hayano, 1986: Sinsabaugh and Linkins, 1987, 1988; Hope and Burns, 1987). CclIulose is a major com~nent of leaf litter. The degradation of crystalline cellulose to glucose is mediated by a group of functionally similar hydrolytic enzymes collectively termed ccllulases. which synergistically interact to hydrolyze crystalline cellulose (Eriksson and Wood. i98.5; Ryu ef ai., 1984). Studies by Sinsabaugh er ui. (1981) and Linkins er ui. (1984) suggested that the mechanisms of cellulose hydrolysis were similar in both terrtstrial and aquatic habitats and were significant in regulating the microbial mineralization of cellulose. However, the interactions between litter and enzymes may be intlucnced by the contrasting hydrologic regimes, resulting in differing patterns of d~om~sition in the two habitats. Our goal was to monitor mass losses and cellulase activities of three decomposing deciduous leaf litters in an upland forest soil and compare the results to those from a similar study by Sinsabaugh er 01. (1981) conducted in a secondorder woodland stream in the same region of western Virginia. Senescent leaves of Cornus @orida L. (flowering dogwood), Acer rubrum L. (red mapfe) and Quercus prinur L. (chestnut oak). were collected in October, at the time of abscission, and air-dried. Nyton litter bags (2mm mesh) containing w 6 g of air-dried leaves were randomfy arrayed along three parallel chains, 4 m in length. pfaced on the forest floor of a second growth oak-maple stand on the south slope of Brush Mountain, near Blacksburg. Virginia. The litter bags were placed directly on the A horizon (pH 4.7. organic content 38%). then covered to a depth of 3-5 cm with the original 0 horizon material. At each sample date, six litter bags of each titter type were recovered. A total of nine samples were retrieved over 680 days beginning I8 May 1983 and ending II April 1985. *To whom all correspondence
should be addressed.
Initial moisture contents were determined by drying litter at 50°C for 24 h, and ash contents by combusting fitter at 45o’C for 8 h. Water-insoluble substances (TAPPI, 1975). and acid-insoluble Iignin contents @Bland, 1977) were also determined for each litter. Sample processing began immediately after collection. The contents of each litter bag were weighed and subsamples were analyxcd for moisture and ash :o calculate mass loss. The remaining litter was pooled by species and homogenized in 50 mr.i acetate buffer. pH 5.0, then centrifuged at lO,OOOgfor IO min. Endocellulase and cxoccllufase were assayed in the pellets and supernatants using carboxymethyl cellulose and mi~r~rystaltinc eellulose, respectively, as substrates (Sinsabaugh and Linkins. 1987). The assays were performed in 50 mMacetate buffer at pti 5 and 21°C Endocellulase activities are reported in arbitrary units based on viscosity changes (Almin and Eriksson, 1967) and exocellulase activities as ~g glucose generated h-i gAFDM_’ (ash-free dry mass). During the first summer (138 days), dogwood litter lost 55.1%. maple 47.2% and oak 24.3% of initial ash-free dry mass (AFDM). After 680 days, the corresponding mass losses were 76.5, 62.6 and 55.5%. This relative mass loss pattern among the three species was consistent throughout the study except during the first sampling period when maple lost more mas than dogwood. These mass loss patterns reflect the differing initial chemical composition of the litters. During the first sampling period, -mass loss may be due largely to leaching (McClaunhertv. 1983). The relative mass losses during the first sampling .period varied with the initial content of water-soluble substances which were 47. t, 42.8 and 40.3% for maple, dogwood and oak, respectively. Differences in long-term mass loss are approximately inversely related to the initial lignin content of the litters (Taylor er ai., I989 and references therein). Dogwood. with the lowest lignin content (3.9%). decomposed more rapidly than maple (10.1%) which decomposed more rapidly than oak litter (19.9%). The same relative patterns of mass loss were found in the lotic system, but the rates were higher. Mass losses quivalent to those seen after 680 days of exposure on the forest floor were recorded after only 154 days of exposure in a woodland stream (Sinsabaugh er al., 1981). In the forest floor system, both endo- and exocellulase activities peaked during the first 30-100 days of exposure (Fig, I), coincident with the period of maximum mass loss. Both enzymes showed similar trends among species. with the
423
4’4
Short
communications
6 6
Tome (days
1
Fig. I. Cellulase activities associated with flowering dogwood. red maple and chestnut oak leaves decomposing on the floor of an oak-maple forest in western Virginia.
highest cellulase activity associated with dogwood. and the least with oak, paralleling the mass loss pattern. In the lotic system. endoccllulase and exocellulase activities were not correlated (Sinsabaugh ef aI.. 1981). The cxoccllulase pattern was similar to that of the terrestrial system. except that activity peaked after only 30 40 days (Fig. 2). However, endoccllulasc activity was markedly dilfercnt. First. peak cndoccllulase activity was temporally displaced from exocellulase. Sozond. the pattern of activity among species was inverted with the greatest activity on oak and the least on dogwood. Thcsc ditl’ercnccs in cellulasc dynamics htwccn the soil and stream systems are reproducible. A repetition of the stream study (R. L. Sinsabaugh. unpublished MS. thesis, University of Virginia, 1980) and subsequent tcrrestrial studies with the same litters (McClaugherty and Linkins. 19X8; Linkins rr ul.. 1990) have yielded the same patterns. We believe that the contrasting patterns of endocellulasc activity on dcyomposing leaf litter in terrestrial and lotic
20
40
60
60
CO
120
140
T~me(doys) Fig. 2. Cellulase activities associated with dogswood. maple and oak leaves decomposing in a second-order woodland stream in western Virginia.
systems can be accounted for by the hydrologic differences of the systems and the differential binding affinities of the cellulase components for litter. In lotic systems. continuous perfusion results in the loss of enzymes that do not strongly associate with insoluble substrates. In the terrestrial system. water content and movement are lower. thus enzyme loss by leachtng is lower. Cellulase have different affinities for litter components. Exocellulases have the highest binding affinity for cellulose (Ryu PI al., 1984). Endocellulases interact more weakly with cellulose and appear to bind preferentially to the ligninhumus fraction of litter (Sinsabaugh and Linkins. 1988). We suggest that in terrestrial and in lotic systems, exocellulases and endocellulases are synthesized concurrently because the degradation of crystalline cellulose requires both types of enzyme. Because exocellulase interacts strongly with cellulose. there are similar activity patterns in both terrestrial and lotic systems. with the quantity of activity related to the amount of accessible cellulose (dogwood > maple > oak). In terrestrial systems, endocellulase activity coincides with exocellulase activity because these enzymes. which interact more weakly with cellulose, are not lost by perfusion as they are in the stream. However. as decomposition proceeds endocellulase immobilization capacity increases because of the relative increases in lignin and humus (Sinsabaugh and Linkins. 1988). The result is that in lotic systems. the apparent peak in endocellulase activity is temporally displaced from the exocellulase, with the quantity of activity varying with the lignin-humus content of the litter (oak > maple > dogwood). In the soil system, endoccllulasc activity declines more rapidly from its maximum, than exocellulase. This dilfcrcncc may be attributable to the dilTcrential stability of the two types of enzymes. Sinsabaugh and Linkins (1989) compared the stability of cellulase components bound to leaf litter and found that exocellulase loses less activity than cndocellulasc in response to wet dry events. A~kncr~l~cl~c,nlenrs -This work was sponsored by the National Science Foundation. Technical assistance was provided by Margot Bridgen.
REFERENCES
Almin K. E. and Eriksson K.-E. (1967) Enzymic degradation of polymers I. Viscometric method for the dctermination of enzymic activity. Biochimico Ed Biophj.sicu Acru 139, 23X -247. Burns R. G. (1983) Extracellular enzyme-substrate interactions in soil. In Microbes in Their Nu~urul Encironmmr (J. H. Slater. R. Whittenbury and J. W. Wimpenny, Eds). pp. 249-298. Cambridge University Press. Effland M. J. (1977) Modified procedure to determine acid-insoluble lignin in wood and pulp. TAPPI 40, 143-144. Eriksson K.-E. and Wood T. M. (1985) Biodegradation of cellulose. In Biosynrhesis und Biodrgrudurion of Wood Componenr.r (T. Higuchi, Ed.). pp. 469-503. Academic Press, New York. Gressel J.. Vered Y.. Bar-Lev S., Milstcin 0. and Flowers H. M. (1983) Partial suppression of cellulose action by artificial lignification of cellulose. Planf Science LPfrers 32, 344-353. Hayano K. (1986) Cellulase complex in tomato field SOII: induction, localization and some properties. Soil Biolog) & Biochemis(rf 18. 215-219. Hope C. F. A. and Burns R. G. (1987) Activity, origins and location of cellulase in a silt loam soil. B10log.r and Ferfiliry of Soils 5. 164-170. Linkins A. E.. Melillo J. M. and Sinsabaugh R. L. (1984) Factors affecting cellulase activity in terrestrial and
Short communications aquatic ecosystems. In Currenf Per5pecrrres in Microbiul Ecology (M. J. Klug and C. A. Reddy. Eds). pp. 572-579. American Society for Microbiology. Wash. Linkins A. E.. Sinsabaugh R. L., McClaugherty C. A. and Melillo J. M. (1990) Cellulase activity on decomposing leaf litter in microcosms. Pbnr cmd Soil (submitted). McClaunhertv C. A. C1983) Soluble oolvohenols and carbohydrates in throughfal) and leaf ht;er decomposition. Acta
Oecologia,‘Oecologia
Generolis
4. 371-385.
McClaugherty C. A. and Linkins A. E. (1988) Extractability of cellulases in forest litter and soil. Biology and Fertility of Soils 6, 322-327. Ryu D.. Kim C. and Mandels M. (1984) Competitive adsorption of cellulase and its significance in a synergistic mechanism. Biotechnology ond Bioengineering 26. 488496.
Sinsabaugh R. L. and Linkins A. E. (1987) Inhibition of
425
the Trichoderma riride cellulase complex by leaf titter extracts. Soil Biology & Biochemistry 19. 719-725. Sinsabaugh R. L. and Linkins A. E. (1988) Adsorption of cellulax components by leaf litter. Soil Bio/ogy dr Biochemistry
20. 927-932.
Sinsabaugh R. L. and Linkins A. E. (1989) Natural disturbances and the activity of Trichodermo riride cellulase complexes. Soil Biology & Biochemistry 21, 835-839. Sinsabaugh R. L.. Benfield E. F. and Linkins A. E. (1981) Cellulase activity associated with the decomposition of leaf litter in a woodland stream. Oikos 36, 184490. TAPPI (1975) Water solubles in wood and pulp (T207 OS-75). Technical Associution of the Pulp and Paper Industry. Allama. Ip. Taylor B. R.. Parkinson D. and Parsons W. J. J. (1989) Nitrogen and lignin content as predictors of litter decay rates: a microcosm test. Ecology 70. 97-104.
0038-0717/90s3.w + 0.00
1990
Copyright c 1990Pcrgamon Press pie
COMPARISON OF CELLULASE ACTIVITY ON DECOMPOSING LEAVES IN A HARDWOOD FOREST AND WOODLAND STREAM A. E. LINKIX and R. L. SINSABAUGH’ Biology apartment,
Clarkson University, Fotsdam, NY 13676, U.S.A.
Biology Department,
Mount Union College, Alliance, OH 44601, U.S.A.
C. A. MCCLAUGHERTY
J. M. MELILLO Tfre Ecosystem Center, Marine Biological Laboratory, Woods Hofe, MA 02543, U.S.A. (Accepted 15 August 1989)
Decomposing leaf litter plays a key role in nutrient cycling and soil organic matter dynamics in forested ecosystems. The initial decomposition of plant fiber is an extracellular process principally mediated by a spectrum of extraceflular enzymes (Burns, 1983). The activity of these enzymes is inffuenced by environmental variables including temperature, moisture and pH as well as the physical and chemical nature of the enzymes and substrates involved (Sinsabaugh er ul., 1981; Gressel et al., l983; Linkins et al., 1984; Hayano, 1986: Sinsabaugh and Linkins, 1987, 1988; Hope and Burns, 1987). CclIulose is a major com~nent of leaf litter. The degradation of crystalline cellulose to glucose is mediated by a group of functionally similar hydrolytic enzymes collectively termed ccllulases. which synergistically interact to hydrolyze crystalline cellulose (Eriksson and Wood. i98.5; Ryu ef ai., 1984). Studies by Sinsabaugh er ui. (1981) and Linkins er ui. (1984) suggested that the mechanisms of cellulose hydrolysis were similar in both terrtstrial and aquatic habitats and were significant in regulating the microbial mineralization of cellulose. However, the interactions between litter and enzymes may be intlucnced by the contrasting hydrologic regimes, resulting in differing patterns of d~om~sition in the two habitats. Our goal was to monitor mass losses and cellulase activities of three decomposing deciduous leaf litters in an upland forest soil and compare the results to those from a similar study by Sinsabaugh er 01. (1981) conducted in a secondorder woodland stream in the same region of western Virginia. Senescent leaves of Cornus @orida L. (flowering dogwood), Acer rubrum L. (red mapfe) and Quercus prinur L. (chestnut oak). were collected in October, at the time of abscission, and air-dried. Nyton litter bags (2mm mesh) containing w 6 g of air-dried leaves were randomfy arrayed along three parallel chains, 4 m in length. pfaced on the forest floor of a second growth oak-maple stand on the south slope of Brush Mountain, near Blacksburg. Virginia. The litter bags were placed directly on the A horizon (pH 4.7. organic content 38%). then covered to a depth of 3-5 cm with the original 0 horizon material. At each sample date, six litter bags of each titter type were recovered. A total of nine samples were retrieved over 680 days beginning I8 May 1983 and ending II April 1985. *To whom all correspondence
should be addressed.
Initial moisture contents were determined by drying litter at 50°C for 24 h, and ash contents by combusting fitter at 45o’C for 8 h. Water-insoluble substances (TAPPI, 1975). and acid-insoluble Iignin contents @Bland, 1977) were also determined for each litter. Sample processing began immediately after collection. The contents of each litter bag were weighed and subsamples were analyxcd for moisture and ash :o calculate mass loss. The remaining litter was pooled by species and homogenized in 50 mr.i acetate buffer. pH 5.0, then centrifuged at lO,OOOgfor IO min. Endocellulase and cxoccllufase were assayed in the pellets and supernatants using carboxymethyl cellulose and mi~r~rystaltinc eellulose, respectively, as substrates (Sinsabaugh and Linkins. 1987). The assays were performed in 50 mMacetate buffer at pti 5 and 21°C Endocellulase activities are reported in arbitrary units based on viscosity changes (Almin and Eriksson, 1967) and exocellulase activities as ~g glucose generated h-i gAFDM_’ (ash-free dry mass). During the first summer (138 days), dogwood litter lost 55.1%. maple 47.2% and oak 24.3% of initial ash-free dry mass (AFDM). After 680 days, the corresponding mass losses were 76.5, 62.6 and 55.5%. This relative mass loss pattern among the three species was consistent throughout the study except during the first sampling period when maple lost more mas than dogwood. These mass loss patterns reflect the differing initial chemical composition of the litters. During the first sampling period, -mass loss may be due largely to leaching (McClaunhertv. 1983). The relative mass losses during the first sampling .period varied with the initial content of water-soluble substances which were 47. t, 42.8 and 40.3% for maple, dogwood and oak, respectively. Differences in long-term mass loss are approximately inversely related to the initial lignin content of the litters (Taylor er ai., I989 and references therein). Dogwood. with the lowest lignin content (3.9%). decomposed more rapidly than maple (10.1%) which decomposed more rapidly than oak litter (19.9%). The same relative patterns of mass loss were found in the lotic system, but the rates were higher. Mass losses quivalent to those seen after 680 days of exposure on the forest floor were recorded after only 154 days of exposure in a woodland stream (Sinsabaugh er al., 1981). In the forest floor system, both endo- and exocellulase activities peaked during the first 30-100 days of exposure (Fig, I), coincident with the period of maximum mass loss. Both enzymes showed similar trends among species. with the
423
4’4
Short
communications
6 6
Tome (days
1
Fig. I. Cellulase activities associated with flowering dogwood. red maple and chestnut oak leaves decomposing on the floor of an oak-maple forest in western Virginia.
highest cellulase activity associated with dogwood. and the least with oak, paralleling the mass loss pattern. In the lotic system. endoccllulase and exocellulase activities were not correlated (Sinsabaugh ef aI.. 1981). The cxoccllulase pattern was similar to that of the terrestrial system. except that activity peaked after only 30 40 days (Fig. 2). However, endoccllulasc activity was markedly dilfercnt. First. peak cndoccllulase activity was temporally displaced from exocellulase. Sozond. the pattern of activity among species was inverted with the greatest activity on oak and the least on dogwood. Thcsc ditl’ercnccs in cellulasc dynamics htwccn the soil and stream systems are reproducible. A repetition of the stream study (R. L. Sinsabaugh. unpublished MS. thesis, University of Virginia, 1980) and subsequent tcrrestrial studies with the same litters (McClaugherty and Linkins. 19X8; Linkins rr ul.. 1990) have yielded the same patterns. We believe that the contrasting patterns of endocellulasc activity on dcyomposing leaf litter in terrestrial and lotic
20
40
60
60
CO
120
140
T~me(doys) Fig. 2. Cellulase activities associated with dogswood. maple and oak leaves decomposing in a second-order woodland stream in western Virginia.
systems can be accounted for by the hydrologic differences of the systems and the differential binding affinities of the cellulase components for litter. In lotic systems. continuous perfusion results in the loss of enzymes that do not strongly associate with insoluble substrates. In the terrestrial system. water content and movement are lower. thus enzyme loss by leachtng is lower. Cellulase have different affinities for litter components. Exocellulases have the highest binding affinity for cellulose (Ryu PI al., 1984). Endocellulases interact more weakly with cellulose and appear to bind preferentially to the ligninhumus fraction of litter (Sinsabaugh and Linkins. 1988). We suggest that in terrestrial and in lotic systems, exocellulases and endocellulases are synthesized concurrently because the degradation of crystalline cellulose requires both types of enzyme. Because exocellulase interacts strongly with cellulose. there are similar activity patterns in both terrestrial and lotic systems. with the quantity of activity related to the amount of accessible cellulose (dogwood > maple > oak). In terrestrial systems, endocellulase activity coincides with exocellulase activity because these enzymes. which interact more weakly with cellulose, are not lost by perfusion as they are in the stream. However. as decomposition proceeds endocellulase immobilization capacity increases because of the relative increases in lignin and humus (Sinsabaugh and Linkins. 1988). The result is that in lotic systems. the apparent peak in endocellulase activity is temporally displaced from the exocellulase, with the quantity of activity varying with the lignin-humus content of the litter (oak > maple > dogwood). In the soil system, endoccllulasc activity declines more rapidly from its maximum, than exocellulase. This dilfcrcncc may be attributable to the dilTcrential stability of the two types of enzymes. Sinsabaugh and Linkins (1989) compared the stability of cellulase components bound to leaf litter and found that exocellulase loses less activity than cndocellulasc in response to wet dry events. A~kncr~l~cl~c,nlenrs -This work was sponsored by the National Science Foundation. Technical assistance was provided by Margot Bridgen.
REFERENCES
Almin K. E. and Eriksson K.-E. (1967) Enzymic degradation of polymers I. Viscometric method for the dctermination of enzymic activity. Biochimico Ed Biophj.sicu Acru 139, 23X -247. Burns R. G. (1983) Extracellular enzyme-substrate interactions in soil. In Microbes in Their Nu~urul Encironmmr (J. H. Slater. R. Whittenbury and J. W. Wimpenny, Eds). pp. 249-298. Cambridge University Press. Effland M. J. (1977) Modified procedure to determine acid-insoluble lignin in wood and pulp. TAPPI 40, 143-144. Eriksson K.-E. and Wood T. M. (1985) Biodegradation of cellulose. In Biosynrhesis und Biodrgrudurion of Wood Componenr.r (T. Higuchi, Ed.). pp. 469-503. Academic Press, New York. Gressel J.. Vered Y.. Bar-Lev S., Milstcin 0. and Flowers H. M. (1983) Partial suppression of cellulose action by artificial lignification of cellulose. Planf Science LPfrers 32, 344-353. Hayano K. (1986) Cellulase complex in tomato field SOII: induction, localization and some properties. Soil Biolog) & Biochemis(rf 18. 215-219. Hope C. F. A. and Burns R. G. (1987) Activity, origins and location of cellulase in a silt loam soil. B10log.r and Ferfiliry of Soils 5. 164-170. Linkins A. E.. Melillo J. M. and Sinsabaugh R. L. (1984) Factors affecting cellulase activity in terrestrial and
Short communications aquatic ecosystems. In Currenf Per5pecrrres in Microbiul Ecology (M. J. Klug and C. A. Reddy. Eds). pp. 572-579. American Society for Microbiology. Wash. Linkins A. E.. Sinsabaugh R. L., McClaugherty C. A. and Melillo J. M. (1990) Cellulase activity on decomposing leaf litter in microcosms. Pbnr cmd Soil (submitted). McClaunhertv C. A. C1983) Soluble oolvohenols and carbohydrates in throughfal) and leaf ht;er decomposition. Acta
Oecologia,‘Oecologia
Generolis
4. 371-385.
McClaugherty C. A. and Linkins A. E. (1988) Extractability of cellulases in forest litter and soil. Biology and Fertility of Soils 6, 322-327. Ryu D.. Kim C. and Mandels M. (1984) Competitive adsorption of cellulase and its significance in a synergistic mechanism. Biotechnology ond Bioengineering 26. 488496.
Sinsabaugh R. L. and Linkins A. E. (1987) Inhibition of
425
the Trichoderma riride cellulase complex by leaf titter extracts. Soil Biology & Biochemistry 19. 719-725. Sinsabaugh R. L. and Linkins A. E. (1988) Adsorption of cellulax components by leaf litter. Soil Bio/ogy dr Biochemistry
20. 927-932.
Sinsabaugh R. L. and Linkins A. E. (1989) Natural disturbances and the activity of Trichodermo riride cellulase complexes. Soil Biology & Biochemistry 21, 835-839. Sinsabaugh R. L.. Benfield E. F. and Linkins A. E. (1981) Cellulase activity associated with the decomposition of leaf litter in a woodland stream. Oikos 36, 184490. TAPPI (1975) Water solubles in wood and pulp (T207 OS-75). Technical Associution of the Pulp and Paper Industry. Allama. Ip. Taylor B. R.. Parkinson D. and Parsons W. J. J. (1989) Nitrogen and lignin content as predictors of litter decay rates: a microcosm test. Ecology 70. 97-104.