This document summarizes a study that examined the effects of simulated nitrogen deposition on oxidative enzyme activity in soil samples from the Gurbantunggut Desert in Northwestern China over two years. The study found that polyphenol oxidase and peroxidase activity exhibited seasonal variations, with higher levels in spring than other seasons, driven mainly by seasonal patterns in soil moisture and temperature. In general, low levels of nitrogen addition (0.5-3 g N m-2 yr-1) had minimal effects on enzyme activity, but higher additions (6-24 g N m-2 yr-1) decreased activity. Few differences were observed between enzyme activity in topsoil and subsoil samples or their responses to nitrogen addition.

1. Temporal dynamics of soil oxidative enzyme activity across a simulated
gradient 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
a b s t r a c ta r t i c l e i n f o
Article history:
Received 22 January 2013
Received in revised form 9 August 2013
Accepted 25 August 2013
Available online 19 September 2013
Keywords:
Nitrogen deposition
Oxidative enzyme
Organic carbon
pH
Soil moisture
Increasing nitrogen (N) deposition has been reported to result in positive, negative, and neutral impacts on soil
oxidative enzyme activities, which are associated with lignin degradation. The oxidative enzyme activities are
usually high and stable in desert ecosystems. Therefore, the exploration of how these enzymes respond to N
addition is critical to evaluate the carbon transformation in desert ecosystems. We present the seasonal variations
in 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 activity
exhibited clear seasonal variations over the 2-year study, with relatively higher values in spring than in the other
seasons. The seasonal changes in oxidative enzyme activity were mainly driven by the seasonal patterns in soil mois-
ture and temperature. Shifts in pH, organic carbon, and electrical conductivity were not closely correlated with the
seasonal changes in oxidative enzyme activity. In general, PPO and peroxidase activity responded minimally to low
N 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 concentrations were 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 two
layers. These results suggest that the carbon transformation related to oxidative enzyme activity is regulated
by 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 global
issues in recent years. The increasing N emitted by industrial and agri-
cultural development has greatly increased the N concentration in the
soil and on leaf surfaces. N deposition increases plant growth, reduces
diversity, and alters ecosystem function (Brooks, 2003; Clark and
Tilman, 2008; Throop, 2005). To further understanding of the above
changes in the individuals and communities, we are interested in the
process by which the results occur in biological communities under in-
creasing N deposition. In general, alterations in soil nutrient transforma-
tion are considered as a key driving force for many other aboveground
changes (Pregitzer et al., 2008). Soil microbial communities, which are
related to nutrient transformation, usually respond earlier and more
sensitively than other communities (Fravolini et al., 2005). Therefore,
an increasing number of studies have focused on soil microbial activity
and its function in soil nutrient transformation.
As a crucial catalyzer in nutrient transformation, microbial enzyme
could promote the rates of degradation. Microbial enzymes involved
in carbon (C), N, and phosphorus (P) transformation respond rapidly
and intensively to N deposition (Enrique et al., 2008; Waldrop and
Zak, 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, and
dissolved 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 “enzymatic
latches” to retain soil organic C in highly managed turf ecosystems
(Yao et al., 2009). Chronic N additions have the potential to directly
modify microbial community composition and function by suppressing
the abundance and activity of fungi which degrade lignin (DeForest
et al., 2004). Although the relationship between the effects of added N
on enzyme activity and decomposition has been questioned by some
studies (Keeler et al., 2009), repression of oxidase activity could be an
important mechanism explaining the negative effects of added N on de-
composition observed in many ecosystems (Carreiro et al., 2000). How-
ever, the factors that regulate phenol oxidase and peroxidase activity
have received limited attention (Sinsabaugh, 2010). Across ecosystems
with divergent soil chemical and physical conditions, differences in
magnitude and effects that oppose oxidase activity have been observed
in response to N addition (Gallo et al., 2004; Stursova et al., 2006;
Waldrop and Zak, 2006; Zeglin et al., 2007). Although the effects of N
addition on oxidative enzyme activity have been studied in different
Geoderma 213 (2014) 261–267
⁎ Corresponding author. Tel.: +86 991 7823158; fax: +86 991 7885320.
E-mail addresses: zhangym@ms.xjb.ac.cn, zhouxb@ms.xjb.ac.cn (Y. Zhang).
0016-7061/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.geoderma.2013.08.030
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journal homepage: www.elsevier.com/locate/geoderma

2. forest and grassland ecosystems, the effects of chronic N addition on ox-
idative enzyme activity in desert ecosystem are unknown, particularly
during different seasons.
Desert soil usually has high oxidative enzyme activity and suffers
from influences of increasing N deposition. The high oxidative enzyme
activity in desert soil may increase the decomposition potential and
limit organic matter accumulation (Stursova and Sinsabaugh, 2008).
In addition, the N deposition in recent years has been high in some
desert areas. It could negatively or positively affect microbial activity,
increase dominance of alien annuals and decrease the diversity of
native 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 nutrient
transformation. The Gurbantunggut Desert is an optimal area for study-
ing oxidative enzyme activity in response to N addition. It is surrounded
by patches of farmland and the natural N deposition into this desert has
increased from 0.5 g N·m−2
·yr−1
in 1991 to 0.8 g N·m−2
·yr−1
in
2006 (Yuan and Wang, 1997; Zhang et al., 2008). Seasonal changes in
soil temperature, moisture, pH, and input N doses could contribute to
changes in oxidative enzyme activity (Gallo et al., 2004; Sinsabaugh,
2010; Sinsabaugh et al., 2005). The soil conditions in the Gurbantunggut
desert are also seasonal, with a high soil water content from snowmelt
and rainfall in spring and low water content in summer and autumn be-
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 chemical
characteristics. Abiotic and biotic characteristics also differ between cer-
tain depths in the Gurbantunggut Desert (Zhang et al., 2005). Organic
matter accumulation, high temperatures, and extreme moisture fluctu-
ations in the Negav Desert produced differences in oxidative enzyme ac-
tivity between the topsoil and deeper soil layers (Alon and Steinberger,
1999). Therefore, monitoring how oxidative enzyme activity responds
to N addition during different seasons at different soil depths will help
us understand how these related nutrient transformations occur and
determine which environmental factors (e.g. moisture, temperature,
pH, etc.) affect them most.
We studied two oxidative enzymes, polyphenol oxidase (PPO) and
peroxidase, (1) to determine the seasonal responses of oxidative
enzyme activity to N addition and their major regulating factors,
(2) to determine the effects of different N application rates on oxidative
enzyme 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 a major driving factor to af-
fect microbial activities (Henry, 2012). Meanwhile, soil depth specific
characteristics 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 seasons
because of the relatively higher soil water content and optimal temper-
ature in spring. (2) Furthermore, the topsoil will have higher PPO and
peroxidase activity and is more sensitive to N addition than the subsoil
because 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 the Gurbantunggut Desert (44.87° N,
87.82° E), Northwestern China. This desert is the second largest in
China, with an area of 48,800 km2
. The mean annual precipitation
ranges from 80 mm to 160 mm, with total precipitation higher in spring
and summer than in autumn and winter. Half of the annual precipita-
tion falls between April and July (47.6%). The mean evaporation rate is
2606.6 mm·yr−1
, thus the soil is water deficient, particularly during
summer. The mean annual air temperature in the region varies between
6 °C to 10 °C, whereas the maximum air temperature exceeds 40 °C. In
winter, about 20 cm of snow covers the soil surface. The desert consists
of massive and dense semifixed sand dunes with scattered shrubs, such
as Haloxylon ammodendron (C. A. Mey) Bunge and H. persicum Bunge ex
Boiss. et Buhse. Annual plants grow extensively, with the highest aver-
age ground coverage of 40% in May. Biological soil crusts are abundant
on the desert surface, where they primarily form and grow during the
moist, cool periods of early spring and autumn.
In October 2008, sixty 8 m × 8 m plots were established on the
interdunes. The plots had similar plant compositions, vegetation cover,
and biological soil crusts on the surface. The plots and treatments were
randomly assigned. The plots were set up 2 m apart to prevent treatment
effects. The study design consists of a control and five N fertilizer treat-
ments, with ten replicates for each treatment. The six treatments received
0, 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 consistent
with 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 N24
are designed to determine the N dose response of plants and microbial
activity under high N treatments, which will help evaluate the potential
effects of higher N deposition (Liu et al., 2011). The fertilizer treatments
were randomly applied onto the 60 plots in two equal doses in March
after the spring snowmelt and in October before the first snowfall of the
winter season. The first treatments began in October 2008, and all the
treatments were applied on the same day. Nitrogen was added as 2:1
NH4
+
: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 water
and sprayed over the plots.
Soil samples were randomly collected from 24 of the 60 plots, with 4
replicates for each treatment (6 N treatments × 4 replicates) on the
first 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 stones with a 2 mm mesh, the soil samples
were packed in ice and taken to the laboratory. The samples were air
dried and used to determine enzyme activity and soil physicochemical
properties (Bai et al., 2009). Soil pH and electrical conductivity (EC)
were measured from a 1:5 mixture of soil and deionized water using a
PHS-3C digital pH meter and a DDS-307A conductivity meter (Precision
and Scientific Corp. Shanghai, China), respectively. Soil organic C con-
tent was measured using the K2Cr2O7 method (Walkley–Black) (Chen
et al., 2007). The average soil temperature and water content during
these months were monitored using a four-parameter soil monitoring
system 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 and
Jastrow, 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 adding
the soil (soil pH was ~8). Soil equivalent to 1 g dry weight was incubated
with 10 mL of 1% PG substrate (79.3 mM) for 2 h at 30 °C. Each soil sam-
ple and control also received 2 mL of 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, which were then extracted with ether. The ab-
sorbance of the extract at 430 nm was measured using a spectrophotom-
eter. PPO and peroxidase activity were standardized by completely
oxidizing a known amount of PG (nmol PG [g · soil]−1
·h−1
) and mea-
suring the absorbance of the reaction products (Guan, 1986). Peroxidase
262 X. Zhou, Y. Zhang / Geoderma 213 (2014) 261–267

3. activity was determined using gross activity from a PG–peroxide assay. A
substrate-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 enzyme activity
were tested by analyzing repeated measurements of a general linear
model. 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). To
determine the seasonal dynamics of oxidative enzyme activity and envi-
ronmental factors, the data from the 12 sampling periods in both 2009
and 2010 were collated into winter (December–February), spring
(March–May), summer (June to August), and autumn (September to
November). Mean values of data from two or three times of sampling
in spring were used. The two-year data on seasonal activity were collat-
ed to compare the N treatment effects on oxidative enzyme activity. A
one-way analysis of variance was used to assess the differences in en-
zyme activity among the N treatments during the four seasons and at
each depth. A Bonferroni t-test was performed on the significant results
to reduce the probability of spurious results. The relationship between
oxidative enzyme activity and environmental factors across the seasons
were analyzed using Pearson's correlation. All statistical analyses were
performed 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 topsoil
and subsoil displayed obvious seasonality (Fig. 1). The soil water con-
tent increased with increasing soil temperature in spring. The tempera-
ture was higher and water content was relatively lower in summer than
in 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-year
study. The topsoil had higher organic C and EC than the subsoil in
both 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 significantly
higher than the subsoil. The average organic C content varied slightly
through the seasons (P N 0.05) except for a significant increase in the
subsoil 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,
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 and
its 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). PPO
activity was higher during spring than during other seasons in both
2009 and 2010 (P b 0.05), with the activity decreasing during summer
and autumn. The topsoil had relatively higher PPO activity than the sub-
soil during most seasons (Table 2; P b 0.05). The peroxidase activity in
the topsoil differed significantly between the two spring seasons
(P b 0.05), but not in the subsoil. The peroxidase activity in both soil
layers 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 as well as between peroxidase activity and
soil moisture in the topsoil (Table 3; P b 0.05). Soil moisture was also
closely correlated with peroxidase activity in the subsoil. Organic C
was strongly correlated with peroxidase activity and with 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 subsoil
during almost all four seasons (Fig. 3A–D). In winter, the PPO activity
was significantly reduced in the N1 treatment (P b 0.05; Fig. 3A). The
PPO activity in the topsoil in the N24 treatment was 21% lower than in
the N0 treatment, with 22% lower in the subsoil. In spring, the effect
of N on PPO activity in the topsoil was not significant between the N0
and the N1 treatments (P N 0.05; Fig. 3B). The N3–N24 treatments sig-
nificantly decreased the PPO activity, with 20% lower activity in the
N24 treatment than in the N0 treatment (P b 0.05). In addition, the
N1–N24 treatments significantly decreased the PPO activity in the sub-
soil, with the lowest activity in the N24 treatment (7% lower than in the
N0 treatment). In summer, PPO activity was insensitive to N addition
unlike in spring. PPO activity only decreased significantly in the N24
treatment (P b 0.05; Fig. 3C), which was 18% lower than in the topsoil
and 13% lower than in the subsoil of the N0 treatment. In autumn, N
addition significantly affected PPO activity in the topsoil and subsoil of
the 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 was
observed in the topsoil during winter (P N 0.05; Fig. 4A). The decrease
was 6% lower in the topsoil and 16% lower in the subsoil of the N24
treatment 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 and
N24 treatments significantly reduced the peroxidase activity in both
soil layers (P b 0.05; Fig. 4C). In autumn, the peroxidase activity in the
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10
-24
-12
0
12
24
36
0
10
20
30
40
50
2010
Temperature(o
C)
Months
T: 5 cm 10 cm
2009
W: 5 cm 10 cm
Watercontent(%)
Fig. 1. Variations in mean daily soil volumetric water content (%, W, bars) and temperature (°C, T, lines) in the topsoil (5 cm) and subsoil (10 cm) from January 2009 to October 2010.
263X. Zhou, Y. Zhang / Geoderma 213 (2014) 261–267

4. N24 treatment significantly decreased only in the topsoil (P b 0.05), and
the effect of N addition was not significant in the subsoil (Fig. 4D).
4. Discussion
4.1. Seasonal changes in oxidative enzyme activity and environmental
factors
PPO and peroxidase activity fluctuated significantly among the sea-
sons. The results were consistent with our hypothesis that oxidative
enzyme activity is highest in spring. Both strong seasonal and no consis-
tent seasonal pattern have been reported by other studies. There was a
strong seasonal pattern in oxidase activity with peaks during spring and
autumn in northern hardwood forests (Sinsabaugh et al., 2005), while
highest oxidase activity occurred in July or August in a Mesic Grassland
(Tiemann and Billings, 2011). However, no consistent seasonal pattern
of variation in phenol oxidase activity was found in soil of a hardwood
forest (Boerner et al., 2005). Understanding the seasonality of enzyme
activity requires understanding the factors that regulate various en-
zyme systems.
Seasonal variations in soil enzyme activity are usually attributed to
differences in temperature pattern and/or water content (Baldrian
et al., 2013; Zwikel et al., 2007). In general, soil moisture manipulations
in field studies have had a much greater influence on potential activity
of extracellular enzyme than warming treatments (Henry, 2012). In
the current study, oxidative enzyme activity fluctuated consistently
for two years. The optimal temperature and high soil water content in
spring probably resulted in high PPO and peroxidase activity. It is be-
cause that enzyme activity in dry plots is more responsive to increases
in soil moisture than that of ambient plots (Steinweg, 2011), and the in-
crease in soil moisture in spring just satisfies the production of enzyme
in desert soil. During the dry season (low soil moisture), the high tem-
peratures in summer and autumn did not induce high oxidative enzyme
activity, possibly because of the deficient soil water content. Thus, the
seasonal variations in oxidative enzyme activity are mainly attributed
to seasonal changes in soil water content, particularly in the topsoil. In-
crease in air temperatures could stimulate peroxidase activity, while not
alter phenol oxidase activity in Sphagnum lawns (Jassey et al., 2012).
Nevertheless, high temperature (N30 °C) can also reduce peroxidase
activity according to an experiment under different incubation temper-
atures (Lang et al., 2000). Although the changes in soil temperature did
not demonstrate a strong influence in both layers, the increased snow-
melt and soil moisture during spring and the soil moisture retained
during other seasons are closely related to shifts in soil temperature.
Therefore, the seasonal patterns of temperature and water content in
desert 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,
and microbial biomass explained much of the variation in enzyme activ-
ity among sites (Keeler et al., 2009). In our study site, significant
changes in pH occurred among the different seasons, with pH remaining
consistent at about 8. However, pH was not significantly correlated with
enzyme activity in the topsoil and subsoil. Thus, the fluctuations in
oxidative enzyme activity should not be attributed to changes in pH in
desert 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 N
amendment 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 associated
with mass loss of litter and particulate organic matter generally increase
with the lignin content and secondary compounds of the material, while
they are not correlated with soil organic matter content (Sinsabaugh,
2010). In the current study, the organic C content in desert soil varied
slightly through the seasons, which is inconsistent with the changes in
oxidative 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 significant
seasonal 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 as
their interactions, determine the seasonal changes in oxidative enzyme
activity in desert soil. Considering C transformation is closely related to
PPO and peroxidase activity, fluctuations in their activity indicate that
lignin degradation, C mineralization, and dissolved organic C export
vary 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 in
the topsoil was not always more sensitive to N addition than in the sub-
soil. These results are inconsistent with our hypothesis that the topsoil
has relatively higher oxidative enzyme activity and is more sensitive
to 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; Kandeler
et al., 2006). The differences are attributed to the changes in microbial
Table 1
Seasonal 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 ± 0.02ab
Spring 8.0 ± 0.02b 115 ± 14a 1.0 ± 0.03a 8.1 ± 0.03ab 106 ± 7a 0.7 ± 0.02b
Summer 7.5 ± 0.04d 108 ± 8a 1.1 ± 0.04a 7.6 ± 0.05d 105 ± 7a 0.7 ± 0.03b
Autumn 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 ± 0.03ab
Spring 7.9 ± 0.02b 125 ± 18a 1.1 ± 0.03a 8.1 ± 0.04ab 110 ± 12a 0.8 ± 0.01a
Summer 8.0 ± 0.02b 124 ± 22a 1.1 ± 0.03a 8.2 ± 0.05a 108 ± 17a 0.8 ± 0.02ab
Autumn 8.0 ± 0.05b 113 ± 23a 1.1 ± 0.02a 8.1 ± 0.04ab 100 ± 14a 0.7 ± 0.01ab
Table 2
Parameters 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** 0.41
Season 7 331.27** 56.52**
Depth × N 5 2.06 0.34
Season × 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.
264 X. Zhou, Y. Zhang / Geoderma 213 (2014) 261–267

5. communities and organic matter accumulation between depths. Al-
though soil depth significantly affected PPO activity, the activity in the
subsoil 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: the
more recalcitrant C compounds, such as lignin-derived aromatic acids
and phenols are preferentially sorbed onto the deeper depth (Kaiser
et al., 2010), thus these compounds may select for microorganisms ca-
pable of producing ligninase enzymes (PPO and peroxidase) to utilize
them in this depth.
4.2. High soil N reduces oxidative enzyme activity
Divergent and even contradictory oxidative enzyme responses to N
addition have been observed in different ecosystems. The significant ef-
fects of N addition are often found in temperate forests. For example, N
addition (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 in
some specific ecosystems, such as sugar maple–basswood forests (Acer
saccharum 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 N
amendment 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, and
soil 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 little
to N addition (10 g N · m−2
·yr−1
), although enzyme activity varies
widely across different grassland ecosystems (Zeglin et al., 2007). The
lack of response suggests that investment in the degradation of recalci-
trant organic matter and lignin in these ecosystems is unaffected by N
addition. Generally, the oxidative enzyme activity in desert grasslands
is less sensitive to N addition than that in forests (Stursova et al.,
2006). In our desert area, the most 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 oxidative
enzyme activity. Therefore, the N added rates may 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 stable
than forest ecosystems (Stursova and Sinsabaugh, 2008). Compared to
northern 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).
100
200
300
400
winter spring summer autumn winter spring summer autumn
winter spring summer autumn winter spring summer autumn
200
300
400
cd
a
ab
d
bc
a
ab
b
D
A
CD
D
C
AB
B
CD
bc
bc
bc
b
c
a
bc
bc
C
AB
BC BC
AB
A
BC ABC
A
2010
PPO(nmolg-1
h-1
)
0-5 cm
5-10 cm
2009
B
Peroxidase(nmolg-1
h-1
)
season
2009 2010
Fig. 2. Seasonal fluctuations in average activity of polyphenol oxidase (PPO; A) and of peroxidase (B) in the topsoil (0–5 cm) and subsoil (5–10 cm) of the N0–N24 treatments. Vertical bars
indicate 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).
Table 3
Pearson's correlation coefficients between oxidative enzyme activity and environmental
factors.
PPO Peroxidase Moisturea
Temperature pH ECb
Organic
C
0–5 cm
PPOc
1 0.43 0.69* 0.34 0.3 −0.06 0.25
Peroxidase 1 0.67* 0.15 0.3 0.3 0.58*
Moisture 1 0.31 0.31 −0.22 0.18
Temperature 1 −0.32 −0.39 0.09
pH 1 0.09 0.17
EC 1 0.69*
Organic C 1
5–10 cm
PPO 1 0.39 0.3 0.14 0.42 −0.33 0.04
Peroxidase 1 0.62* 0.09 0.16 −0.25 0.14
Moisture 1 0.46 0.23 −0.49 0.35
Temperature 1 −0.15 −0.71 −0.2
pH 1 −0.32 0.17
EC 1 0.25
Organic C 1
a
* = P b 0.05.
b
EC = electrical conductivity.
c
PPO = polyphenol oxidase.
265X. Zhou, Y. Zhang / Geoderma 213 (2014) 261–267

6. In the present study, if the activity unit was calculated base on organic
matter (OM), the PPO activity would fall into the same range of study
by Stursova and Sinsabaugh (2008). The high oxidative enzyme activity
could increase the decomposition potential and limits soil organic
matter accumulation (Stursova and Sinsabaugh, 2008). However, N
addition decreased PPO and peroxidase activity, especially at high N
concentrations. Because peroxidase can oxidize lignin macromolecules
into simple phenols (Tien and Kirk, 1983), and phenol oxidize the ben-
zene ring in a phenolic compound (Hammel, 1997), an excess of added
N would promote incomplete lignin degradation and potentially the
accumulation of soil organic matter. Thus, the monitoring of PPO and
peroxidase activity in this desert may present a suitable and forward in-
dicator on C cycle, especially under increasing deposition.
However, a meta-analysis found that N enrichment had no significant
effect on litter decay when averaged across studies, because this process
depended on interaction between fertilization rate, site-specific ambient
N-deposition level, and litter quality (Knorr et al., 2005). In addition, pho-
tochemical oxidation, which is not restricted to periods of high water
availability, can supplement enzymatic oxidation and increase organic
matter decomposition in arid ecosystems (Gallo et al., 2009). Therefore,
although the C transformation rate would be altered by N addition, we
could not determine whether the decrease in oxidase activity in the
a
a
ab
ab
bc bc
cd c
cd
c
d c
a
a
ab
ab
ab
bc
bc
c
c
bc
d
c
a aab aab a
ab ab
ab ab
b b a aa aab abcd abbc abd b
0
100
200
300
400
PPO(nmolg-1h-1)
N0 N0.5 N1
N3 N6 N24
A
0
100
200
300
400 B
0
100
200
300
400
Depth
C
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). Vertical
bars indicate standard errors of the mean (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).
a
ab
a
a
a
bca
bc
a
bc
a c
a a
a
aa
a
ab
aab
ab a
a aa aab abab abb bc b
a aa aab a
ab a
ab
a
b
a
0
100
200
300
400
peroxidase(nmolg-1
h-1
)
N0 N0.5 N1
N3 N6 N24
A
0
100
200
300
400 B
0
100
200
300
400
Depth
C
0-5 cm 5-10 cm
0-5 cm 5-10 cm
0-5 cm 5-10 cm
0-5 cm 5-10 cm
0
100
200
300
400
Depth
D
Fig. 4. Seasonal changes in soil peroxidase activity in the topsoil (0–5 cm) and subsoil (5–10 cm) under six N treatments (A: winter; B: spring; C: summer; D: autumn). Vertical bars
indicate standard errors of the mean (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

7. high N treatments certainly promotes organic matter accumulation in de-
sert soils. In summary, much work needs on the effects of N addition on
litter 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 soil
water content and temperature. N addition reduces PPO and peroxidase
activity, particularly at high levels (i.e., treatments N6 and N24). Mini-
mal significant differences in oxidative enzyme activity and sensitivity
to N addition were observed between in the topsoil and the subsoil. Al-
though oxidative enzyme activity decreased, we could not determine
whether suppressing oxidative enzyme activity leads to organic matter
accumulation.
Acknowledgments
This work was jointly funded by grants from the National Natural
Science Foundation of China (Grant no. 41001181, U1203301). Two
anonymous reviewers provide helpful feedback that improves the
manuscript. We thank Wu Shuo, Chen Xi, Liu Xing, and the many
other students of Xinjiang Agricultural University for their many hours
of soil nutrient and enzyme activity analyses.
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