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

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.

本研究旨在探究限制或促进温带森林土壤中蛋白酶（proteolytic enzymes）活性与合成量的过程。为达成该研究目标，我们开展了一系列整合观测与实验，以验证蛋白酶活性的概念模型：即蛋白酶活性由土壤温度、湿度、底物浓度以及酶库大小这四个参数的交互作用共同决定。我们选取4种优势温带森林树种作为模式系统，这些树种的土壤有机质（Soil Organic Matter, SOM）化学特征及其真菌共生体的酶学功能均存在差异。4个树种的菌根共生类型各不相同：白蜡树（*Fraxinus americana*）与糖枫（*Acer saccharum*）形成丛枝菌根（arbuscular mycorrhizal, AM），而东部铁杉（*Tsuga canadensis*）与美洲山毛榉（*Fagus grandifolia*）则形成外生菌根（ectomycorrhizal, ECM）。此外，与外生菌根共生的树种，其叶片凋落物与土壤有机质的碳氮比（C:N）高于丛枝菌根共生树种。
土壤样本采集自两个样地，分别为哈佛大学森林保护区的Prospect Hill区域与皮斯加州立森林公园。两处样地的土地利用历史与林分年龄均相近。两地土壤均为源自花岗岩-片岩-片麻岩基岩上冰碛物发育的典型弱育湿润始成土（Typic Dystrochrepts，隶属于始成土纲Inceptisols）。每个样地均设置了以4种目标树种之一为优势种的实验样方：糖枫与美洲山毛榉林分位于皮斯加样地，东部铁杉与白蜡树林分位于哈佛大学森林样地。后续又在Prospect Hill区域的红栎（*Quercus rubra*）林分中，于MES塔影响范围内增设了样方。每个样地内均设置6个重复样方，样方半径为8米，为单优群落样方，需满足以下3项标准：(1) 立木胸高断面积占比80%以上为目标树种；(2) 新鲜凋落物层以目标树种为主；(3) 样方核心5米区域内仅生长目标树种。仅山毛榉与铁杉样地存在显著的有机土层，白蜡树样地缺乏有机土层，这可能与蚯蚓的存在有关——其余样地均未发现蚯蚓。
2008年，我们探究了温度与底物可用性对温带森林土壤蛋白酶活性的影响，以及与外生菌根或丛枝菌根共生的树种之间，蛋白酶与其他氮获取相关酶（即几丁质酶与木质素酶）的活性差异。我们采用生长季内3个时间点采集的土壤样本开展析因室内实验，分别在两种温度（采样时的原位温度与23℃）与两种底物水平（天然状态或添加酪蛋白形式的外源蛋白）下测定蛋白酶活性。此外，我们还开展了野外原位根系生长实验，以比较外生菌根与丛枝菌根树种的根系对酶产量与活性的影响差异。2009年，我们测定了生长季内3个时间点下蛋白酶的温度敏感性：从所有4个树种的土壤样本中，在0~35℃共6个温度梯度下定量检测了蛋白酶活性。2010年，我们测定了所有4个树种根际与非根际土壤中蛋白酶、几丁质酶与木质素酶的活性。
研究结果表明，蛋白酶活性受底物的限制作用强于温度，且随着生长季内土壤温度升高，两种限制作用均有所减弱。细根可通过促进微生物酶的合成，刺激根际（根系紧邻区域）内的蛋白酶活性。外生菌根根系对蛋白酶活性的刺激作用强于丛枝菌根根系。本研究结果显示，在底物可用性未提升的前提下，气候变暖对温带森林土壤蛋白酶活性的影响较为有限。此外，改变树木地下碳分配的全球变化事件，对外生菌根林分的氮循环的影响可能大于丛枝菌根林分。