Geoderma 213 (2014) 261–267

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Temporal dynamics of soil oxidative enzyme activity across a simulatedgradient of nitrogen deposition in the Gurbantunggut Desert,Northwestern China

Xiaobing Zhou, Yuanming Zhang ⁎Xinjiang Institute of Ecology and Geography, Key Laboratory of Biogeography and Bioresource in Arid Land, Chinese Academy of Sciences, Urumqi 830011, China

⁎ Corresponding author. Tel.: +86 991 7823158; fax: +E-mail addresses: zhangym@ms.xjb.ac.cn, zhouxb@ms

0016-7061/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.geoderma.2013.08.030

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 January 2013Received in revised form 9 August 2013Accepted 25 August 2013Available online 19 September 2013

Keywords:Nitrogen depositionOxidative enzymeOrganic carbonpHSoil moisture

Increasing nitrogen (N) deposition has been reported to result in positive, negative, and neutral impacts on soiloxidative enzyme activities, which are associated with lignin degradation. The oxidative enzyme activities areusually high and stable in desert ecosystems. Therefore, the exploration of how these enzymes respond to Naddition is critical to evaluate the carbon transformation indesert ecosystems.We present the seasonal variationsin oxidative enzyme activity and their responses to simulated N deposition in the topsoil (0–5 cm) and in subsoil(5–10 cm) of the Gurbantunggut Desert in Northwestern China. Polyphenol oxidase (PPO) and peroxidase activityexhibited clear seasonal variations over the 2-year study, with relatively higher values in spring than in the otherseasons. The seasonal changes in oxidative enzymeactivityweremainly drivenby the seasonal patterns in soilmois-ture and temperature. Shifts in pH, organic carbon, and electrical conductivity were not closely correlated with theseasonal changes in oxidative enzyme activity. In general, PPO and peroxidase activity respondedminimally to lowN addition (0.5–3 g N m−2 yr−1), but the activity decreased in response to high N addition (6–24 g N m−2 yr−1).Although higher organic carbon concentrationswere observed in the topsoil than in the subsoil, few significant dif-ferences in PPO and peroxidase activity and in their sensitivity to N addition were observed between the twolayers. These results suggest that the carbon transformation related to oxidative enzyme activity is regulatedby the seasonal patterns of soil moisture, soil temperature, and the N concentration in desert soil.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Nitrogen (N) deposition has become one of the most heated globalissues in recent years. The increasing N emitted by industrial and agri-cultural development has greatly increased the N concentration in thesoil and on leaf surfaces. N deposition increases plant growth, reducesdiversity, and alters ecosystem function (Brooks, 2003; Clark andTilman, 2008; Throop, 2005). To further understanding of the abovechanges in the individuals and communities, we are interested in theprocess by which the results occur in biological communities under in-creasingNdeposition. In general, alterations in soil nutrient transforma-tion are considered as a key driving force for many other abovegroundchanges (Pregitzer et al., 2008). Soil microbial communities, which arerelated to nutrient transformation, usually respond earlier and moresensitively than other communities (Fravolini et al., 2005). Therefore,an increasing number of studies have focused on soil microbial activityand its function in soil nutrient transformation.

As a crucial catalyzer in nutrient transformation, microbial enzymecould promote the rates of degradation. Microbial enzymes involvedin carbon (C), N, and phosphorus (P) transformation respond rapidly

86 991 7885320..xjb.ac.cn (Y. Zhang).

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and intensively to N deposition (Enrique et al., 2008; Waldrop andZak, 2006; Zeglin et al., 2007). For example, oxidative enzymes, includ-ing phenol oxidase and peroxidase, mediate many key ecosystem func-tions, such as lignin degradation, humification, C mineralization, anddissolved organic C export (Grandy et al., 2008; Waldrop and Zak,2006). Soil phenol oxidase regulates the activity of hydrolytic soil en-zymes by controlling soluble phenolics and function as “enzymaticlatches” to retain soil organic C in highly managed turf ecosystems(Yao et al., 2009). Chronic N additions have the potential to directlymodify microbial community composition and function by suppressingthe abundance and activity of fungi which degrade lignin (DeForestet al., 2004). Although the relationship between the effects of added Non enzyme activity and decomposition has been questioned by somestudies (Keeler et al., 2009), repression of oxidase activity could be animportantmechanism explaining the negative effects of added N on de-composition observed inmany ecosystems (Carreiro et al., 2000). How-ever, the factors that regulate phenol oxidase and peroxidase activityhave received limited attention (Sinsabaugh, 2010). Across ecosystemswith divergent soil chemical and physical conditions, differences inmagnitude and effects that oppose oxidase activity have been observedin response to N addition (Gallo et al., 2004; Stursova et al., 2006;Waldrop and Zak, 2006; Zeglin et al., 2007). Although the effects of Naddition on oxidative enzyme activity have been studied in different

262 X. Zhou, Y. Zhang / Geoderma 213 (2014) 261–267

forest and grassland ecosystems, the effects of chronic N addition on ox-idative enzyme activity in desert ecosystem are unknown, particularlyduring different seasons.

Desert soil usually has high oxidative enzyme activity and suffersfrom influences of increasing N deposition. The high oxidative enzymeactivity in desert soil may increase the decomposition potential andlimit organic matter accumulation (Stursova and Sinsabaugh, 2008).In addition, the N deposition in recent years has been high in somedesert areas. It could negatively or positively affect microbial activity,increase dominance of alien annuals and decrease the diversity ofnative annual plants (Brooks, 2003; McCrackin et al., 2008). Therefore,the ways by which oxidative enzymes in desert soil respond to N addi-tion should be examined in terms of their essential role in nutrienttransformation. The Gurbantunggut Desert is an optimal area for study-ing oxidative enzyme activity in response toN addition. It is surroundedby patches of farmland and the natural N deposition into this desert hasincreased from 0.5 g N·m−2·yr−1 in 1991 to 0.8 g N·m−2·yr−1 in2006 (Yuan and Wang, 1997; Zhang et al., 2008). Seasonal changes insoil temperature, moisture, pH, and input N doses could contribute tochanges in oxidative enzyme activity (Gallo et al., 2004; Sinsabaugh,2010; Sinsabaugh et al., 2005). The soil conditions in theGurbantunggutdesert are also seasonal, with a high soil water content from snowmeltand rainfall in spring and lowwater content in summer and autumnbe-cause of high soil temperatures. These soil changes affect the germina-tion and growth of ephemeral plants and annuals (Wang et al., 2006),thereby causing shifts in organic matter content and other soil chemicalcharacteristics. Abiotic and biotic characteristics also differ between cer-tain depths in the Gurbantunggut Desert (Zhang et al., 2005). Organicmatter accumulation, high temperatures, and extrememoisture fluctu-ations in theNegavDesert produced differences in oxidative enzymeac-tivity between the topsoil and deeper soil layers (Alon and Steinberger,1999). Therefore, monitoring how oxidative enzyme activity respondsto N addition during different seasons at different soil depths will helpus understand how these related nutrient transformations occur anddetermine which environmental factors (e.g. moisture, temperature,pH, etc.) affect them most.

We studied two oxidative enzymes, polyphenol oxidase (PPO) andperoxidase, (1) to determine the seasonal responses of oxidativeenzyme activity to N addition and their major regulating factors,(2) to determine the effects of different N application rates on oxidativeenzyme activity in desert soil, and (3) to evaluate the effects of N addi-tion on oxidative activity between the topsoil (0–5 cm) and the subsoil(5–10 cm). Soil moisture was considered as amajor driving factor to af-fect microbial activities (Henry, 2012). Meanwhile, soil depth specificcharacteristics might lead to the differences in responding magnitude(Alon and Steinberger, 1999). Therefore, we hypothesize that (1) oxida-tive enzyme activity is higher during spring than during other seasonsbecause of the relatively higher soil water content and optimal temper-ature in spring. (2) Furthermore, the topsoil will have higher PPO andperoxidase activity and is more sensitive to N addition than the subsoilbecause of its higher organic matter content (Zhang et al., 2005).

2. Materials and methods

2.1. Study site and sample collection

The study site is in the center of theGurbantunggut Desert (44.87°N,87.82° E), Northwestern China. This desert is the second largest inChina, with an area of 48,800 km2. The mean annual precipitationranges from80 mmto 160 mm,with total precipitation higher in springand summer than in autumn and winter. Half of the annual precipita-tion falls between April and July (47.6%). The mean evaporation rate is2606.6 mm·yr−1, thus the soil is water deficient, particularly duringsummer. Themean annual air temperature in the region varies between6 °C to 10 °C, whereas the maximum air temperature exceeds 40 °C. Inwinter, about 20 cm of snow covers the soil surface. The desert consists

of massive and dense semifixed sand dunes with scattered shrubs, suchasHaloxylon ammodendron (C. A. Mey) Bunge andH. persicum Bunge exBoiss. et Buhse. Annual plants grow extensively, with the highest aver-age ground coverage of 40% in May. Biological soil crusts are abundanton the desert surface, where they primarily form and grow during themoist, cool periods of early spring and autumn.

In October 2008, sixty 8 m × 8 m plots were established on theinterdunes. The plots had similar plant compositions, vegetation cover,and biological soil crusts on the surface. The plots and treatments wererandomly assigned. The plots were set up 2 m apart to prevent treatmenteffects. The study design consists of a control and five N fertilizer treat-ments,with ten replicates for each treatment. The six treatments received0, 0.5, 1.0, 3.0, 6.0, and 24.0 g N·m−2·yr−1, and denoted as N0, N0.5, N1,N3, N6, and N24, respectively. The N0.5 to N1 treatments are consistentwith the natural N deposition in adjacent desert areas, whereas N3 is sim-ilar to the N deposition in the Mojave Desert (Brooks, 2003). N6 and N24are designed to determine the N dose response of plants and microbialactivity under high N treatments, which will help evaluate the potentialeffects of higher N deposition (Liu et al., 2011). The fertilizer treatmentswere randomly applied onto the 60 plots in two equal doses in Marchafter the spring snowmelt and in October before the first snowfall of thewinter season. The first treatments began in October 2008, and all thetreatments were applied on the same day. Nitrogen was added as 2:1NH4

+:NO3− (NH4NO3 and NH4Cl), which approximates the N deposition

in Urumqi City near the desert (Zhang et al., 2008). For uniform dispersal,the N amendments for each treatment were dissolved in 30 L of waterand sprayed over the plots.

Soil sampleswere randomly collected from 24 of the 60 plots, with 4replicates for each treatment (6 N treatments × 4 replicates) on thefirst sampling date. Samples were collected from the same four plots af-terward. The soil samples were collected 12 times from the topsoil(0–5 cm) and the subsoil (5–10 cm) in 2009 (February, March, May,July, October, and December) and 2010 (April, May, June, July, Septem-ber, and October). The sampling dates were based on the seasonal cli-mate conditions and the phenology of plant growth.

Three cores were collected along a diagonal line in each plot, pooled,and then thoroughly mixed to represent spatial heterogeneity. After re-moving plant roots and large stoneswith a 2 mmmesh, the soil sampleswere packed in ice and taken to the laboratory. The samples were airdried and used to determine enzyme activity and soil physicochemicalproperties (Bai et al., 2009). Soil pH and electrical conductivity (EC)were measured from a 1:5 mixture of soil and deionized water using aPHS-3C digital pHmeter and a DDS-307A conductivitymeter (Precisionand Scientific Corp. Shanghai, China), respectively. Soil organic C con-tent was measured using the K2Cr2O7 method (Walkley–Black) (Chenet al., 2007). The average soil temperature and water content duringthese months were monitored using a four-parameter soil monitoringsystem installed near the plots (Channel Corp., Beijing, China).

2.2. Oxidative enzyme activity

Oxidative enzyme activity was determined as previously reported(Zhou et al., 2012). PPO (EC 1.10.3.2) and peroxidase (EC 1.11.1.7) activ-ity were assayed using pyrogallol (PG) as the substrate (Allison andJastrow, 2006). Considering the oxidation rate of PG depends on pH,the PG substrate solution was prepared using phosphate buffer(pH 4.5) to avoid rapid PG autoxidation in alkaline solution after addingthe soil (soil pHwas ~8). Soil equivalent to 1 g dryweight was incubatedwith 10 mL of 1% PG substrate (79.3 mM) for 2 h at 30 °C. Each soil sam-ple and control also received 2 mLof 0.3%H2O2 before incubation for per-oxidase assays. After incubation, 2.5 mL of phosphate buffer (pH 4.5)was added to the samples,whichwere then extractedwith ether. The ab-sorbance of the extract at 430 nmwasmeasured using a spectrophotom-eter. PPO and peroxidase activity were standardized by completelyoxidizing a known amount of PG (nmol PG [g · soil]−1·h−1) and mea-suring the absorbance of the reaction products (Guan, 1986). Peroxidase

263X. Zhou, Y. Zhang / Geoderma 213 (2014) 261–267

activity was determined using gross activity from a PG–peroxide assay. Asubstrate-free control and a soil-free were included during the incuba-tion of each enzyme.

2.3. Statistical analyses

The effects of N treatments and seasons on oxidative enzymeactivitywere tested by analyzing repeated measurements of a general linearmodel. Data analysis was performed based upon a split-plot design,with soil depth as the split-plot factor. The models included all main ef-fects and interactions of depth × N, season × N, season × depth (N). Todetermine the seasonal dynamics of oxidative enzymeactivity and envi-ronmental factors, the data from the 12 sampling periods in both 2009and 2010 were collated into winter (December–February), spring(March–May), summer (June to August), and autumn (September toNovember). Mean values of data from two or three times of samplingin springwere used. The two-year data on seasonal activity were collat-ed to compare the N treatment effects on oxidative enzyme activity. Aone-way analysis of variance was used to assess the differences in en-zyme activity among the N treatments during the four seasons and ateach depth. A Bonferroni t-test was performed on the significant resultsto reduce the probability of spurious results. The relationship betweenoxidative enzyme activity and environmental factors across the seasonswere analyzed using Pearson's correlation. All statistical analyses wereperformed using SAS V8 software (SAS Institute Inc., Cary, NC, U.S.A.)at the P = 0.05 level.

3. Results

3.1. Seasonal changes in environmental condition

The soil temperature and volumetric water content in the topsoiland subsoil displayed obvious seasonality (Fig. 1). The soil water con-tent increased with increasing soil temperature in spring. The tempera-turewas higher andwater contentwas relatively lower in summer thanin other seasons. The soil pH in both the topsoil and subsoil was approx-imately 8.0 (Table 1). Although pH differed significantly across the sea-sons (P b 0.05), no consistent trend was observed during the two-yearstudy. The topsoil had higher organic C and EC than the subsoil inboth 2009 and 2010. However, similar to pH, no obvious trends or sig-nificant differences in EC were observed between 2009 and 2010(P N 0.05). The average organic C content in the topsoil was significantlyhigher than the subsoil. The average organic C content varied slightlythrough the seasons (P N 0.05) except for a significant increase in thesubsoil in the spring of 2010.

3.2. Seasonal effects on oxidative enzyme activities

The season, depth, N, and their interactions except for depth × N,significantly affected polyphenol oxidase activity (Table 2). The season,

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N application rate, the interaction of season with depth (N) and of sea-son with N affected peroxidase activity (P b 0.05). However, depth andits interaction with N did not significantly influence peroxidase activity(P N 0.05).

PPO and peroxidase activity in both the topsoil and subsoil fluctuat-ed with the seasons in 2009 and 2010 (Fig. 2A and B; P b 0.05). PPOactivity was higher during spring than during other seasons in both2009 and 2010 (P b 0.05), with the activity decreasing during summerand autumn. The topsoil had relatively higher PPO activity than the sub-soil during most seasons (Table 2; P b 0.05). The peroxidase activity inthe topsoil differed significantly between the two spring seasons(P b 0.05), but not in the subsoil. The peroxidase activity in both soillayers differed slightly among summer, autumn, and winter (P N 0.05).

Pearson's correlation analysis revealed a significant relationship be-tween PPO and soil moisture aswell as between peroxidase activity andsoil moisture in the topsoil (Table 3; P b 0.05). Soil moisture was alsoclosely correlated with peroxidase activity in the subsoil. Organic Cwas strongly correlatedwith peroxidase activity andwith EC in the sub-soil, but no relationships were observed among the other factors.

3.3. Effects of N addition during different seasons

N addition reduced the PPO activity in both the upper and subsoilduring almost all four seasons (Fig. 3A–D). In winter, the PPO activitywas significantly reduced in the N1 treatment (P b 0.05; Fig. 3A). ThePPO activity in the topsoil in the N24 treatment was 21% lower than inthe N0 treatment, with 22% lower in the subsoil. In spring, the effectof N on PPO activity in the topsoil was not significant between the N0and the N1 treatments (P N 0.05; Fig. 3B). The N3–N24 treatments sig-nificantly decreased the PPO activity, with 20% lower activity in theN24 treatment than in the N0 treatment (P b 0.05). In addition, theN1–N24 treatments significantly decreased the PPO activity in the sub-soil, with the lowest activity in the N24 treatment (7% lower than in theN0 treatment). In summer, PPO activity was insensitive to N additionunlike in spring. PPO activity only decreased significantly in the N24treatment (P b 0.05; Fig. 3C), which was 18% lower than in the topsoiland 13% lower than in the subsoil of the N0 treatment. In autumn, Naddition significantly affected PPO activity in the topsoil and subsoil ofthe N3–N24 treatments compared with the N0 treatment (P b 0.05;Fig. 3D).

Peroxidase activity showed a similar trend to PPO activity after N ad-dition (Fig. 4A–D). No significant decrease in peroxidase activity wasobserved in the topsoil during winter (P N 0.05; Fig. 4A). The decreasewas 6% lower in the topsoil and 16% lower in the subsoil of the N24treatment compared with the N0 treatment. In spring, only the peroxi-dase activity in the topsoil was significantly affected in the N24 treat-ment (P b 0.05), not in the subsoil (Fig. 4B). In summer, the N6 andN24 treatments significantly reduced the peroxidase activity in bothsoil layers (P b 0.05; Fig. 4C). In autumn, the peroxidase activity in the

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C, T, lines) in the topsoil (5 cm) and subsoil (10 cm) from January 2009 to October 2010.

Table 1Seasonal changes in pH, electrical conductivity (EC), and organic C in the topsoil (0–5 cm) and subsoil (5–10 cm) (mean ± SE, n = 6; P b 0.05; Bonferroni's test).

Year Season 0–5 cm 5–10 cm

pH EC(μs·cm−1)

Organic C(g·kg−1)

pH EC(μs·cm−1)

Organic C(g·kg−1)

2009 Winter 8.2 ± 0.03a 136 ± 22a 1.1 ± 0.03a 8.3 ± 0.09a 124 ± 19a 0.7 ± 0.02abSpring 8.0 ± 0.02b 115 ± 14a 1.0 ± 0.03a 8.1 ± 0.03ab 106 ± 7a 0.7 ± 0.02bSummer 7.5 ± 0.04d 108 ± 8a 1.1 ± 0.04a 7.6 ± 0.05d 105 ± 7a 0.7 ± 0.03bAutumn 7.7 ± 0.01c 122 ± 7a 1.1 ± 0.04a 7.8 ± 0.07cd 117 ± 7a 0.7 ± 0.05ab

2010 Winter 7.7 ± 0.04c 154 ± 14a 1.2 ± 0.04a 7.8 ± 0.04bc 136 ± 14a 0.8 ± 0.03abSpring 7.9 ± 0.02b 125 ± 18a 1.1 ± 0.03a 8.1 ± 0.04ab 110 ± 12a 0.8 ± 0.01aSummer 8.0 ± 0.02b 124 ± 22a 1.1 ± 0.03a 8.2 ± 0.05a 108 ± 17a 0.8 ± 0.02abAutumn 8.0 ± 0.05b 113 ± 23a 1.1 ± 0.02a 8.1 ± 0.04ab 100 ± 14a 0.7 ± 0.01ab

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N24 treatment significantly decreased only in the topsoil (P b 0.05), andthe effect of N addition was not significant in the subsoil (Fig. 4D).

4. Discussion

4.1. Seasonal changes in oxidative enzyme activity and environmentalfactors

PPO and peroxidase activity fluctuated significantly among the sea-sons. The results were consistent with our hypothesis that oxidativeenzyme activity is highest in spring. Both strong seasonal and no consis-tent seasonal pattern have been reported by other studies. There was astrong seasonal pattern in oxidase activity with peaks during spring andautumn in northern hardwood forests (Sinsabaugh et al., 2005), whilehighest oxidase activity occurred in July or August in a Mesic Grassland(Tiemann and Billings, 2011). However, no consistent seasonal patternof variation in phenol oxidase activity was found in soil of a hardwoodforest (Boerner et al., 2005). Understanding the seasonality of enzymeactivity requires understanding the factors that regulate various en-zyme systems.

Seasonal variations in soil enzyme activity are usually attributed todifferences in temperature pattern and/or water content (Baldrianet al., 2013; Zwikel et al., 2007). In general, soil moisture manipulationsin field studies have had a much greater influence on potential activityof extracellular enzyme than warming treatments (Henry, 2012). Inthe current study, oxidative enzyme activity fluctuated consistentlyfor two years. The optimal temperature and high soil water content inspring probably resulted in high PPO and peroxidase activity. It is be-cause that enzyme activity in dry plots is more responsive to increasesin soil moisture than that of ambient plots (Steinweg, 2011), and the in-crease in soil moisture in spring just satisfies the production of enzymein desert soil. During the dry season (low soil moisture), the high tem-peratures in summer and autumndid not induce high oxidative enzymeactivity, possibly because of the deficient soil water content. Thus, theseasonal variations in oxidative enzyme activity are mainly attributedto seasonal changes in soil water content, particularly in the topsoil. In-crease in air temperatures could stimulate peroxidase activity, while notalter phenol oxidase activity in Sphagnum lawns (Jassey et al., 2012).Nevertheless, high temperature (N30 °C) can also reduce peroxidase

Table 2Parameters regulating the activities of PPO and peroxidase using General Linear Model.

Source df F-valuea

PPOb Peroxidase

N 5 57.55** 25.23**Depth 1 30.43** 0.41Season 7 331.27** 56.52**Depth × N 5 2.06 0.34Season × N 35 3.62** 1.54*Season × depth (N) 42 8.68** 4.26**

a * = P b 0.05. ** = P b 0.01.b PPO = polyphenol oxidase.

activity according to an experiment under different incubation temper-atures (Lang et al., 2000). Although the changes in soil temperature didnot demonstrate a strong influence in both layers, the increased snow-melt and soil moisture during spring and the soil moisture retainedduring other seasons are closely related to shifts in soil temperature.Therefore, the seasonal patterns of temperature and water content indesert soil affect PPO and peroxidase activity.

Aside from soil water content and temperature, pH is another essen-tial abiotic factor that affects phenol oxidase and peroxidase activity,with a generally positive relationship between pH and oxidative en-zyme activity (Sinsabaugh, 2010). Differences in pH, moisture, soil C,andmicrobial biomass explainedmuch of the variation in enzyme activ-ity among sites (Keeler et al., 2009). In our study site, significantchanges in pHoccurred among thedifferent seasons,with pH remainingconsistent at about 8. However, pHwas not significantly correlatedwithenzyme activity in the topsoil and subsoil. Thus, the fluctuations inoxidative enzyme activity should not be attributed to changes in pH indesert soil.

Soil organic matter plays an essential role in the responses of oxida-tive enzyme activity to environmental change in some ecosystems. o-Diphenol oxidase activity increases with increasing organic C and Namendment in arid climates, but only up to a certain level; thereafter,it plateaus or even diminishes (Bastida et al., 2008). Across ecosystems,potential activities of phenol oxidase and peroxidase which associatedwithmass loss of litter and particulate organicmatter generally increasewith the lignin content and secondary compounds of thematerial, whilethey are not correlated with soil organic matter content (Sinsabaugh,2010). In the current study, the organic C content in desert soil variedslightly through the seasons, which is inconsistent with the changes inoxidative enzyme activity. Correlation analysis also revealed the mini-mal effects of soil organic C on the seasonal changes in oxidative activity.By contrast, even though Quercus-dominated forests have significantseasonal changes in organic C, they have no consistent seasonal varia-tion in phenol oxidase activity (Boerner et al., 2005).

The contribution of soil water content and temperature, as well astheir interactions, determine the seasonal changes in oxidative enzymeactivity in desert soil. Considering C transformation is closely related toPPO and peroxidase activity, fluctuations in their activity indicate thatlignin degradation, C mineralization, and dissolved organic C exportvary with the seasonal dynamics of soil water content and temperature.For instance, in forest ecosystems in Michigan, increased oxidative en-zyme activity is often associated with reduced dissolved organic C(Waldrop and Zak, 2006).

Peroxidase activity was not significantly different between the top-soil and subsoil, (Table 2). In addition, the oxidative enzyme activity inthe topsoil was not alwaysmore sensitive to N addition than in the sub-soil. These results are inconsistent with our hypothesis that the topsoilhas relatively higher oxidative enzyme activity and is more sensitiveto N addition than the subsoil because of its higher organic C content.Many studies have documented differences in microbial activity at dif-ferent soil depths (Enrique et al., 2008; Gallo et al., 2004; Kandeleret al., 2006). The differences are attributed to the changes in microbial

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Fig. 2. Seasonalfluctuations in average activity of polyphenol oxidase (PPO;A) and of peroxidase (B) in the topsoil (0–5 cm) and subsoil (5–10 cm) of theN0–N24 treatments. Vertical barsindicate standard errors of the mean (n = 6 replicates). Different capital and lowercase letters represent significant differences among the different seasons in the topsoil and subsoil(P b 0.05; Bonferroni's test).

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communities and organic matter accumulation between depths. Al-though soil depth significantly affected PPO activity, the activity in thesubsoil was generally higher than that in the topsoil (Fig. 2A). This phe-nomenon of higher activity in subsoil was similar to study of Daradick(2007) in forests. The underlying mechanism might be as follows: themore recalcitrant C compounds, such as lignin-derived aromatic acidsand phenols are preferentially sorbed onto the deeper depth (Kaiseret al., 2010), thus these compounds may select for microorganisms ca-pable of producing ligninase enzymes (PPO and peroxidase) to utilizethem in this depth.

Table 3Pearson's correlation coefficients between oxidative enzyme activity and environmentalfactors.

PPO Peroxidase Moisturea Temperature pH ECb OrganicC

0–5 cmPPOc 1 0.43 0.69* 0.34 0.3 −0.06 0.25Peroxidase 1 0.67* 0.15 0.3 0.3 0.58*Moisture 1 0.31 0.31 −0.22 0.18Temperature 1 −0.32 −0.39 0.09pH 1 0.09 0.17EC 1 0.69*Organic C 1

5–10 cmPPO 1 0.39 0.3 0.14 0.42 −0.33 0.04Peroxidase 1 0.62* 0.09 0.16 −0.25 0.14Moisture 1 0.46 0.23 −0.49 0.35Temperature 1 −0.15 −0.71 −0.2pH 1 −0.32 0.17EC 1 0.25Organic C 1

a * = P b 0.05.b EC = electrical conductivity.c PPO = polyphenol oxidase.

4.2. High soil N reduces oxidative enzyme activity

Divergent and even contradictory oxidative enzyme responses to Naddition have been observed in different ecosystems. The significant ef-fects of N addition are often found in temperate forests. For example, Naddition (3 and 8 g N·m−2·yr−1) decreases phenol oxidase and perox-idase activity in northern temperate forests (Gallo et al., 2004).While insome specific ecosystems, such as sugar maple–basswood forests (Acersaccharum Marsh.; Tilia americana L.), increased soil N significantly in-creases phenol oxidase activity (Waldrop and Zak, 2006). In addition,phenol oxidase activity dropped in soil, but increased in litter after Namendment in an A. saccharum forest soil (Saiya-Cork et al., 2002).The changes in oxidative enzyme activity in these ecosystems are relat-ed to shifts in the soil microbial community, decomposition rates, andsoil C accumulation (Sinsabaugh, 2010). N addition could alter the pro-duction and biochemical composition of plant litter, and in turn, sub-strate availability for microbial community (DeForest et al., 2004).However, the oxidative enzyme activity in grasslands responds littleto N addition (10 g N · m−2·yr−1), although enzyme activity varieswidely across different grassland ecosystems (Zeglin et al., 2007). Thelack of response suggests that investment in the degradation of recalci-trant organic matter and lignin in these ecosystems is unaffected by Naddition. Generally, the oxidative enzyme activity in desert grasslandsis less sensitive to N addition than that in forests (Stursova et al.,2006). In our desert area, themost significant decreases in PPO and per-oxidase activity occurred only in the high N treatments (N6–N24) dur-ing most seasons. Low N additions (i.e., treatments N0.5–N1 for PPO,and N0.5–N3 for peroxidase) had minimal negative effects on oxidativeenzyme activity. Therefore, theN added ratesmay be an important driv-ing factor for the significant N effects on oxidative enzyme activity.

The oxidative enzyme activity in desert grasslands is high and stablethan forest ecosystems (Stursova and Sinsabaugh, 2008). Compared tonorthern temperate forest, the activity of phenol oxidase and peroxi-dase in desert soil was 100 to 3200 times and 4 to 16 times higher,respectively (Sinsabaugh et al., 2005; Stursova and Sinsabaugh, 2008).

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0-5 cm 5-10 cm 0-5 cm 5-10 cm

0-5 cm 5-10 cm0-5 cm 5-10 cm

0

100

200

300

400 D

Fig. 3. Seasonal changes in polyphenol oxidase (PPO) activity in the topsoil (0–5 cm) and subsoil (5–10 cm) under six N treatments (A:winter; B: spring; C: summer; D: autumn). Verticalbars indicate standard errors of themean (n = 4 replicates). Different letters represent significant differences among the different N application rates in the topsoil and subsoil (P b 0.05;Bonferroni's test).

266 X. Zhou, Y. Zhang / Geoderma 213 (2014) 261–267

In the present study, if the activity unit was calculated base on organicmatter (OM), the PPO activity would fall into the same range of studyby Stursova and Sinsabaugh (2008). The high oxidative enzyme activitycould increase the decomposition potential and limits soil organicmatter accumulation (Stursova and Sinsabaugh, 2008). However, Naddition decreased PPO and peroxidase activity, especially at high Nconcentrations. Because peroxidase can oxidize lignin macromoleculesinto simple phenols (Tien and Kirk, 1983), and phenol oxidize the ben-zene ring in a phenolic compound (Hammel, 1997), an excess of addedN would promote incomplete lignin degradation and potentially theaccumulation of soil organic matter. Thus, the monitoring of PPO and

aab

aa

abca

bca

bca c

a aa aab abab abb bc b

0

100

200

300

400

pero

xida

se (

nmol

g-1

h-1

)

N0 N0.5 N1 N3 N6 N24

A

0

100

200

300

400 C

0-5 cm 5-10 cm

0-5 cm 5-10 cm

Fig. 4. Seasonal changes in soil peroxidase activity in the topsoil (0–5 cm) and subsoil (5–10indicate standard errors of the mean (n = 4 replicates). Different letters represent significantBonferroni's test).

peroxidase activity in this desertmay present a suitable and forward in-dicator on C cycle, especially under increasing deposition.

However, ameta-analysis found that N enrichment had no significanteffect on litter decay when averaged across studies, because this processdepended on interaction between fertilization rate, site-specific ambientN-deposition level, and litter quality (Knorr et al., 2005). In addition, pho-tochemical oxidation, which is not restricted to periods of high wateravailability, can supplement enzymatic oxidation and increase organicmatter decomposition in arid ecosystems (Gallo et al., 2009). Therefore,although the C transformation rate would be altered by N addition, wecould not determine whether the decrease in oxidase activity in the

a aa

aaa

abaab ab a

a aa aab aab a

aba

ba

0

100

200

300

400 B

Depth

0-5 cm 5-10 cm

0-5 cm 5-10 cm

0

100

200

300

400

Depth

D

cm) under six N treatments (A: winter; B: spring; C: summer; D: autumn). Vertical barsdifferences among the different N application rates in the topsoil and subsoil (P b 0.05;

267X. Zhou, Y. Zhang / Geoderma 213 (2014) 261–267

highN treatments certainly promotes organicmatter accumulation in de-sert soils. In summary, much work needs on the effects of N addition onlitter degradation and on its relationship with oxidase.

5. Conclusions

PPO and peroxidase activity fluctuate with the season. These fluctu-ations in activity are probably driven by the seasonal changes in soilwater content and temperature. N addition reduces PPO and peroxidaseactivity, particularly at high levels (i.e., treatments N6 and N24). Mini-mal significant differences in oxidative enzyme activity and sensitivityto N addition were observed between in the topsoil and the subsoil. Al-though oxidative enzyme activity decreased, we could not determinewhether suppressing oxidative enzyme activity leads to organic matteraccumulation.

Acknowledgments

This work was jointly funded by grants from the National NaturalScience Foundation of China (Grant no. 41001181, U1203301). Twoanonymous reviewers provide helpful feedback that improves themanuscript. We thank Wu Shuo, Chen Xi, Liu Xing, and the manyother students of Xinjiang Agricultural University for their many hoursof soil nutrient and enzyme activity analyses.

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