Harvard Forest

Data Archive

HF180

Proteolytic Enzyme Activity in Temperate Forest Soils at Harvard Forest and Pisgah State Forest 2007-2010

Related Publications

Data

• hf180-01: temperature and substrate limitation to proteolytic activity (preview)
• hf180-02: in-growth experiment (preview)
• hf180-03: enzyme activity (preview)
• hf180-04: temperature sensitivity of proteolytic enzymes (preview)
• hf180-05: rhizosphere vs. bulk soil (preview)
• hf180-06: root biomass (preview)
• hf180-07: soil carbon and nitrogen (preview)
• hf180-08: litterfall (preview)

Overview

• Lead: Edward Brzostek, Adrien Finzi
• Investigators: John Drake
• Contact: Information Manager
• Start date: 2007
• End date: 2010
• Status: complete
• Location: Prospect Hill Tract (Harvard Forest), Pisgah State Forest (NH)
• Latitude: +42.50 to +42.87 degrees
• Longitude: -72.45 to -72.18 degrees
• Elevation:
• Datum: WGS84
• Taxa: Acer saccharum (sugar maple), Fagus grandifolia (american beech), Fraxinus americana (white ash), Tsuga canadensis (eastern hemlock)
• Release date: 2023
• Language: English
• EML file: knb-lter-hfr.180.10
• DOI: digital object identifier
• EDI: data package
• DataONE: data package
• Related links:
• Extracellular Enzyme Activity in Rhizosphere Soil at Harvard Forest and Pisgah State Forest 2010
• Study type: short-term measurement
• Research topic: soil carbon and nitrogen dynamics
• LTER core area: organic matter movement
• Keywords: biomass, carbon, litterfall, mycorrhizae, nitrogen, rhizosphere, soil organic matter
• Abstract:

  The objective of this research is to investigate the processes that limit or promote the activity and production of proteolytic enzymes in temperate forest soils. To meet this objective, we performed a series of integrated observations and experiments to investigate a conceptual model of proteolytic enzyme activity whereby activity is a function of the interaction between four parameters: soil temperature and moisture, substrate concentration, and the enzyme pool size. We used four dominant temperate forest tree species that differ in SOM chemistry and the enzymatic capabilities of their fungal symbionts as a model system. These four species differed in mycorrhizal association, with white ash (Fraxinus americana) and sugar maple (Acer saccharum) supporting arbuscular mycorrhizal (AM) fungi and eastern hemlock (Tsuga canadensis) and American beech (Fagus grandifolia) supporting ectomycorrhizal (ECM) fungi. Further, the ECM associated species have leaf litter and SOM that is characterized by higher ratios of C:N than the AM associated.

  Soil samples were collected two sites, one located at the Prospect Hill Tract of the Harvard Forest and the other at the Pisgah State Forest. The sites have similar land use history and stand age. Soils at both sites are inceptisols classified as Typic Dystrochrepts derived from glacial till overlying granite-schist-gneiss bedrock. Experimental plots dominated by one of four target tree species were established at each site. Stands of sugar maple (Acer saccharum) and American beech (Fagus grandifolia) were located in Pisgah. Stands of Eastern hemlock (Tsuga canadensis) and white ash (Fraxinus americana) were located in Harvard Forest. At a later date, plots were also established in a red oak (Quercus rubra) stand on Prospect Hill in the MES tower footprint. At each site we located six replicate, 8 m radius, monodominant plots that were based on the following criteria: (1) more than 80% of the standing basal area was composed of the target tree species, (2) the fresh litterfall layer was dominated by the target species, and (3) the core 5 m of the plot contained only the target tree species. Significant organic horizons were present only in the beech and hemlock plots. The lack of organic horizon in the white ash plots may be due to the presence of earthworms, which are not present in the other plots.

  In 2008, we investigated how temperature and substrate availability impact the activity of proteolytic enzymes in temperate forest soils, and whether the activity of proteolytic enzymes and other enzymes involved in the acquisition of N (i.e., chitinolytic and ligninolytic enzymes) differs between trees species that form associations with either ECM or AM fungi. We performed a factorial lab experiment using soils sampled at three time points over the growing season, where we assayed proteolytic rates at two temperatures (field temperature at time of sampling and 23 deg C) and two substrate levels (ambient or elevated protein in the form of casein). Further, we performed an in-growth experiment in the field to investigate differences between ECM and AM tree roots in their effects on enzyme production and activity. In 2009, we assayed the temperature sensitivity of proteolytic enzymes at three time points over the growing season. We quantified proteolytic enzyme activity at six temperatures ranging from 0-35 deg C in the soils from all four species. Finally, in 2010, we assayed the activity of proteolytic, chitinolytic, and ligninolytic enzymes in the rhizosphere and bulk soil of all four tree species.

  We found that proteolytic activity was more limited by substrate than by temperature, with declines in both limitations as soil temperature increased over the growing season. Fine roots stimulated proteolytic activity in the rhizosphere, the zone immediately around the root, likely by enhancing microbial enzyme production. Ectomycorrhizal roots stimulated activity more than arbuscular mycorrhizal roots. The results of this research suggest that climate warming in the absence of increases in substrate availability will have a modest effect on proteolytic activity in temperate forests. Further, global changes that alter belowground carbon allocation by trees are likely to have a larger impact on N cycling in ectomycorrhizal stands than in arbuscular mycorrhizal stands.

• Methods:

  Site Description

  Study plots dominated by one of four target tree species were established at each site. Stands of sugar maple (Acer saccharum) and American beech (Fagus grandifolia) were located in the Pisgah State Forest, NH. Stands of eastern hemlock (Tsuga canadensis) and white ash (Fraxinus americana) were located in the Harvard Forest. These four species differed in mycorrhizal association, with white ash and sugar maple supporting arbuscular mycorrhizal (AM) fungi and hemlock and beech supporting ectomycorrhizal (ECM) fungi. At each site we located six replicate, 8-m radius, monodominant plots where the target tree species constituted 100% of standing basal area in the inner 5-m core and more than 80% in the entire 8-m radius plot.

  Soil samples were collected in 2008. At each sampling point, we collected a 10 X 20 cm sample of the Oe and Oa horizon in each of the hemlock and beech plots. Directly beneath this location, we removed the top 15 cm of mineral soil using a 4.7-cm diameter soil bulk-density sampler. Only mineral soil samples were collected in the maple and ash plots since the organic horizon was non-existent. After collection, roots were removed from the soils and divided between fine (less than 2mm diameter) and coarse (greater than 2mm) roots. Soils were subsequently sieved through a 2-mm mesh for mineral soils and an 8-mm mesh for organic horizon. Roots were washed and both roots and soils were oven-dried at 60°C for at least 48 hours. Soils were later analyzed for C and N content on an element analyzer (NC 2500 Elemental Analyzer, CE Elantech, Lakewood, NJ, USA).

  Litterfall was collected in two litter baskets per plot and sorted to species, dried at 60°C, and weighed. Litterfall was collected three times per year, in October and November, and litter weights from the three collections were added to compute annual litterfall.

  Temperature and Substrate Limitation to Proteolytic Activity

  In 2008, we performed an experiment to determine how temperature and substrate availability affect proteolytic enzyme activity throughout the growing season. In this factorial experiment, proteolytic rates were assayed at two temperatures (field temperature at time of sampling and 23 deg C) and two substrate levels (ambient or elevated protein in the form of casein) in soils from each plot and both soil horizons (n=144). This experiment was performed for three sample dates in 2008. The temperature of the soil in the field in April, June, and August were 4 deg C, 15 deg C, and 18 deg C. Within 12 hours of soil sample collection four, replicate 2-3g subsamples of each soil were placed in their proper incubation temperature and allowed to equilibrate in closed 50ml centrifuge tubes for 12 hours. After equilibration, we assayed proteolysis. To perform the assay under ambient protein conditions, initial and incubated subsamples received 10ml of a 0.5mM sodium acetate buffer (pH 5.0) with a small volume of toluene (400µl) added to inhibit microbial uptake. The assay of proteolysis under elevated conditions was performed similarly, except the sodium acetate buffer contained 0.6% casein. After the reagent addition, the initial samples were immediately terminated and the incubated subsamples were placed back into their respective assay temperature for 4 hours. Proteolytic rates for each experimental treatment were calculated from the difference between amino acid concentrations in the incubated and initial subsamples of each treatment assayed using the OPAME method.

  In-growth Experiment

  We conducted a growing season long field experiment to investigate the role of roots on proteolysis and extracellular enzyme activity. This experiment used mineral soil in-growth cores and organic horizon ingrowth bags. Organic horizon bags (5cm x 10cm x 3cm) and PVC core frames for mineral soil (5cm diameter x 15cm deep) were constructed using either 1mm fiberglass mesh or 50µm nylon mesh (Sefar Industries, Depew, NY) (Langley et al. 2006). The 50µm nylon mesh excludes roots but allows fungal hyphae and heterotrophic microbes to explore the soil, while the 1mm mesh allows both roots and fungal hyphae ingrowth. The in-growth cores were filled with soil collected from each plot in April of 2008. The organic and mineral soil horizons were sieved as previously described. From these samples we created a single, bulk sample of organic or mineral soil horizon for each species. The homogenized soil was then placed back into the in-growth treatments to approximate bulk density. In May of 2008, one core from each treatment was placed into each plot and soil horizon (n=48 mineral soil cores; n=24 organic horizon bags). In-growth bags were installed at the interface between the organic horizon and mineral soil horizon layers. In-growth cores were installed into the soil by first removing the organic horizon, then placing the core in the top 15cm of the mineral soil horizon and finally replacing the organic horizon. The cores/bags were harvested during the first week of October 2008 and then assayed for proteolytic, N-acetyl-glucosaminidase (NAG), and phenol oxidase enzyme activity. NAG and phenol oxidase activity were assayed using standard fluorometric and colorometric methods, respectively.

  Extracellular Enzyme Activity

  At each sample date that proteolytic enzyme activity was assayed in 2008 and 2009, we also assayed the activity of NAG, phenol oxidase, and acid phosphatase in the mineral soil and organic horizon of each plot.

  Temperature Sensitivity of Proteolytic Enzymes

  We collected soil samples in April, June, and August of 2009 from each of the six replicate plots for each species. Proteolytic rates in the top 15 cm of soil were assayed in the lab at ambient substrate levels and at six temperatures - 4 deg C, 10 deg C, 17.5 deg C, 23 deg C, 30 deg C, and 35 deg C. Four, replicate 2-3g subsamples of soil from each sample were placed in their proper incubation temperature and allowed to equilibrate for 12 hours. After equilibration, we assayed proteolytic enzyme activity at the target incubation temperature as described above.

  Rhizosphere vs. Bulk Soil

  Soil samples were collected in May, June, and August of 2010 from each of the six replicate plots for each species. In the laboratory, we separated the mineral soil horizons into rhizosphere and bulk soil fractions. Fine roots were removed from each sample. Soil adhering to fine roots was operational classified as rhizosphere soil. The remaining soil was defined as the bulk soil fraction. In both soil fractions, we assayed the concentration of amino acids and the activity of proteolytic enzymes, NAG, acid phosphatase, phenol oxidase, and peroxidase.

• Organization: Harvard Forest. 324 North Main Street, Petersham, MA 01366, USA. Phone (978) 724-3302. Fax (978) 724-3595.

• Project: The Harvard Forest Long-Term Ecological Research (LTER) program examines ecological dynamics in the New England region resulting from natural disturbances, environmental change, and human impacts. (ROR).

• Funding: National Science Foundation LTER grants: DEB-8811764, DEB-9411975, DEB-0080592, DEB-0620443, DEB-1237491, DEB-1832210.

• Use: This dataset is released to the public under Creative Commons CC0 1.0 (No Rights Reserved). Please keep the dataset creators informed of any plans to use the dataset. Consultation with the original investigators is strongly encouraged. Publications and data products that make use of the dataset should include proper acknowledgement.

• License: Creative Commons Zero v1.0 Universal (CC0-1.0)

• Citation: Brzostek E, Finzi A. 2023. Proteolytic Enzyme Activity in Temperate Forest Soils at Harvard Forest and Pisgah State Forest 2007-2010. Harvard Forest Data Archive: HF180 (v.10). Environmental Data Initiative: https://doi.org/10.6073/pasta/11067d8e647af066056cf4e4ac8b3736.

Detailed Metadata

hf180-01: temperature and substrate limitation to proteolytic activity

1. date: date
2. year: year
3. doy: day of year soils sampled (unit: nominalDay / missing value: NA)
4. species: species
   • ACSA: sugar maple (Acer saccharum)
   • FAGR: American beech (Fagus grandifolia)
   • FRAM: white ash (Fraxinus americana)
   • TSCA: eastern hemlock (Tsuga canadensis)
5. assay.temp: temperature in °C at which proteolysis was assayed in the lab (unit: celsius / missing value: NA)
6. substrate: assay substrate conditions
   • ambient: no protein added
   • elevated: protein added
7. plot: plot number
8. horizon: horizon
   • MS: top 15cm of mineral soil
   • OH: organic horizon; Note: OH were only present in TSCA and FAGR plots.
9. proteolysis: proteolysis, the rate of protein depolymerization into free amino acids (converted to μg amino-acid-N/g dry soil/per hour) (unit: microgramPerGramPerHour / missing value: NA)

hf180-02: in-growth experiment

1. date: date
2. year: year
3. doy: day of year soils sampled (unit: nominalDay / missing value: NA)
4. species: species
   • ACSA: sugar maple (Acer saccharum)
   • FAGR: American beech (Fagus grandifolia)
   • FRAM: white ash (Fraxinus americana)
   • TSCA: eastern hemlock (Tsuga canadensis)
5. plot: plot number
6. fungi: mycorrhizal fungal association of each tree species
   • am: arbuscular mycorrhizal
   • ecm: ectomycorrhizal
7. horizon: horizon
   • MS: top 15cm of mineral soil
   • OH: organic horizon
8. treatment: treatment
   • No Roots: roots excluded from ingrowth core or bag
   • Root: root ingrowth allowed
9. proteolyis: proteolysis, the rate of protein depolymerization into free amino acids (converted to μg amino-acid-N/g dry soil/per hour) (unit: microgramPerGramPerHour / missing value: NA)
10. nag: N-acetyl-glucosaminidase activity. Units: µmol g dry soil-1 hr-1 (unit: micromolePerGramPerHour / missing value: NA)
11. phenox: Phenol Oxidase activity. Units: µmol g dry soil-1 hr-1 (unit: micromolePerGramPerHour / missing value: NA)

hf180-03: enzyme activity

1. date: date
2. year: year
3. doy: day of year soils sampled (unit: nominalDay / missing value: NA)
4. species: species
   • ACSA: sugar maple (Acer saccharum)
   • FAGR: American beech (Fagus grandifolia)
   • FRAM: white ash (Fraxinus americana)
   • TSCA: eastern hemlock (Tsuga canadensis)
5. plot: plot number
6. fungi: mycorrhizal fungal association of each tree species
   • am: arbuscular mycorrhizal
   • ecm: ectomycorrhizal
7. horizon: horizon
   • MS: top 15cm of mineral soil
   • OH: organic horizon
8. nag: N-acetyl-glucosaminidase activity. Units: µmol g dry soil-1 hr-1 (unit: micromolePerGramPerHour / missing value: NA)
9. ap: acid phosphatase activity. Units: µmol g dry soil-1 hr-1 (unit: micromolePerGramPerHour / missing value: NA)
10. phenox: Phenol Oxidase Activity. Units: µmol g dry soil-1 hr-1 (unit: micromolePerGramPerHour / missing value: NA)

hf180-04: temperature sensitivity of proteolytic enzymes

1. date: date
2. year: year
3. doy: day of year soils sampled (unit: nominalDay / missing value: NA)
4. species: species
   • ACSA: sugar maple (Acer saccharum)
   • FAGR: American beech (Fagus grandifolia)
   • FRAM: white ash (Fraxinus americana)
   • TSCA: eastern hemlock (Tsuga canadensis)
5. plot: plot number
6. fungi: mycorrhizal fungal association of each tree species
   • am: arbuscular mycorrhizal
   • ecm: ectomycorrhizal
7. horizon: horizon
   • MS: top 15cm of mineral soil
   • OH: organic horizon
8. pro4: proteolysis (converted to μg amino-acid-N/g dry soil/per hour) assayed at 4°C in the lab (unit: microgramPerGramPerHour / missing value: NA)
9. pro10: proteolysis (converted to μg amino-acid-N/g dry soil/per hour) assayed at 10°C in the lab (unit: microgramPerGramPerHour / missing value: NA)
10. pro17.5: proteolysis (converted to μg amino-acid-N/g dry soil/per hour) assayed at 17.5°C in the lab (unit: microgramPerGramPerHour / missing value: NA)
11. pro23: proteolysis (converted to μg amino-acid-N/g dry soil/per hour) assayed at 23°C in the lab (unit: microgramPerGramPerHour / missing value: NA)
12. pro30: proteolysis (converted to μg amino-acid-N/g dry soil/per hour) assayed at 30°C in the lab (unit: microgramPerGramPerHour / missing value: NA)
13. pro35: proteolysis (converted to μg amino-acid-N/g dry soil/per hour) assayed at 35°C in the lab (unit: microgramPerGramPerHour / missing value: NA)

hf180-05: rhizosphere vs. bulk soil

1. date: date
2. year: year
3. doy: day of year soils sampled (unit: nominalDay / missing value: NA)
4. species: species
   • ACSA: sugar maple (Acer saccharum)
   • FAGR: American beech (Fagus grandifolia)
   • FRAM: white ash (Fraxinus americana)
   • TSCA: eastern hemlock (Tsuga canadensis)
5. plot: plot number
6. soil.type: soil type
   • B: bulk soil
   • R: rhizosphere soil
   • OH: organic horizon
7. proteolysis: proteolysis (converted to μg amino-acid-N/g dry soil/per hour) (unit: microgramPerGramPerHour / missing value: NA)
8. aa: concentration of amino acids. Units: ug Amino Acid-Nitrogen g dry soil-1 (unit: microgramPerGram / missing value: NA)
9. nag: N-acetyl-glucosaminidase activity. Units: µmol g dry soil-1 hr-1 (unit: micromolePerGramPerHour / missing value: NA)
10. ap: acid phosphatase activity. Units: µmol g dry soil-1 hr-1 (unit: micromolePerGramPerHour / missing value: NA)
11. phenox: Phenol Oxidase activity. Units: µmol g dry soil-1 hr-1 (unit: micromolePerGramPerHour / missing value: NA)
12. perox: Peroxidase activity. Units: µmol g dry soil-1 hr-1 (unit: micromolePerGramPerHour / missing value: NA)

hf180-06: root biomass

1. year: year of measurement
2. plot: plot number
3. site: site
   • Pisgah: Pisgah State Forest
   • ash: Prospect Hill - ash
   • hemlock: Prospect Hill - hemlock
4. species: tree species
   • ACSA: sugar maple
   • FAGR: American beech
   • FRAM: white ash
   • TSCA: eastern hemlock
5. roots: whether fine roots (less than 2mm diameter) or coarse roots (greater than 2 mm diameter)
6. horizon: whether organic or mineral soil horizon
7. mass: root mass (g roots) (unit: gram / missing value: NA)
8. mass.m2: root mass per unit surface area (g roots/m2) (unit: gramsPerSquareMeter / missing value: NA)

hf180-07: soil carbon and nitrogen

1. year: year of measurement
2. month: month of measurement
3. site: site
   • Pisgah: Pisgah State Forest
   • ash: Prospect Hill - ash
   • hemlock: Prospect Hill - hemlock
   • ems: Prospect Hill - ems
4. species: tree species
   • ACSA: sugar maple
   • FAGR: American beech
   • FRAM: white ash
   • TSCA: eastern hemlock
   • QURU: red oak
5. horizon: whether organic or mineral soil horizon
6. c: soil carbon content (%) (unit: dimensionless / missing value: NA)
7. n: soil nitrogen content (%) (unit: dimensionless / missing value: NA)

hf180-08: litterfall

1. site: site
   • Pisgah: Pisgah State Forest
   • Ash: Prospect Hill - ash
   • Hemlock: Prospect Hill - hemlock
2. species: tree species
   • ASCA: sugar maple
   • FAGR: American beech
   • FRAM: white ash
   • TSCA: eastern hemlock
3. plot: plot number
4. basket: litter basket number
5. collection.1: litter mass (dry litter) for collection date #1 – Oct 13, 2007 (unit: gram / missing value: NA)
6. collection.2: litter mass (dry litter) for collection date #2 – Nov 12, 2007 (unit: gram / missing value: NA)
7. collection.3: litter mass (dry litter) for collection date #3 – Nov 30, 2007 (unit: gram / missing value: NA)
8. total.2007: total litterfall mass per surface area in 2007 (unit: gramsPerSquareMeter / missing value: NA)
9. collection.4: litter mass (dry litter) for collection date #4 – Oct 8, 2008 (unit: gram / missing value: NA)
10. collection.5: litter mass for collection date #5 – Oct 21, 2008 (unit: gram / missing value: NA)
11. collection.6: litter mass for collection date #6 – Nov 9, 2008 (unit: gram / missing value: NA)
12. total.2008: total litterfall mass per surface area in 2008 (unit: gramsPerSquareMeter / missing value: NA)