enhanced root exudation stimulates soil nitrogen transformations in a subalpine coniferous forest...

Enhanced root exudation stimulates soil nitrogentransformations in a subalpine coniferous forest underexperimental warmingHUAJUN Y IN * † , YUFE I L I * † , J UAN X IAO * † , ZHENFENG XU ‡ , X INY IN CHENG* † and

QING LIU*†

*Chengdu Institute of Biology, Chinese Academy of Sciences, P. O. Box 416, Chengdu 610041, China, †Key Laboratory of

Mountain Ecological Restoration and Bioresource Utilization, Ecological Restoration Biodiversity Conservation Key Laboratory of

Sichuan Province, Chinese Academy of Sciences, Chengdu 610041, China, ‡Institute of Ecology & Forestry, Sichuan Agricultural

University, Chengdu 611130, China

Abstract

Despite the perceived importance of exudation to forest ecosystem function, few studies have attempted to examine

the effects of elevated temperature and nutrition availability on the rates of root exudation and associated microbial

processes. In this study, we performed an experiment in which in situ exudates were collected from Picea asperata

seedlings that were transplanted in disturbed soils exposed to two levels of temperature (ambient temperature and

infrared heater warming) and two nitrogen levels (unfertilized and 25 g N m�2 a�1). Here, we show that the trees

exposed to an elevated temperature increased their exudation rates I (lg C g�1 root biomass h�1), II (lg C cm�1 root

length h�1) and III (lg C cm�2 root area h�1) in the unfertilized plots. The altered morphological and physiological

traits of the roots exposed to experimental warming could be responsible for this variation in root exudation. More-

over, these increases in root-derived C were positively correlated with the microbial release of extracellular enzymes

involved in the breakdown of organic N (R2 = 0.790; P = 0.038), which was coupled with stimulated microbial activ-

ity and accelerated N transformations in the unfertilized soils. In contrast, the trees exposed to both experimental

warming and N fertilization did not show increased exudation rates or soil enzyme activity, indicating that the stimu-

latory effects of experimental warming on root exudation depend on soil fertility. Collectively, our results provide

preliminary evidence that an increase in the release of root exudates into the soil may be an important physiological

adjustment by which the sustained growth responses of plants to experimental warming may be maintained via

enhanced soil microbial activity and soil N transformation. Accordingly, the underlying mechanisms by which plant

root-microbe interactions influence soil organic matter decomposition and N cycling should be incorporated into

climate-carbon cycle models to determine reliable estimates of long-term C storage in forests.

Keywords: exudation, N transformation, nutrient availability, subalpine coniferous forest, warming

Received 7 January 2013 and accepted 24 January 2013

Introduction

The boreal forest has been indicated as one of the ter-

restrial ecosystems that may have a larger sink strength

than expected, but uncertainty regarding the persis-

tence of the sink has hindered efforts to predict biotic

feedback to climate change (Lindroth et al., 1998).

Numerous studies have reported that an elevated tem-

perature increases tree seedling carbon (C) assimilation

rates (Saxe et al., 2001; Wang et al., 2003), plant growth

and biomass accumulation (Xu & Juma, 1994; Zhao &

Liu, 2009) and forest net primary productivity (NPP)

(Scheller & Mladenoff, 2005; Hudson & Henry, 2009).

Nutrient availability, mainly N, is the primary limiting

factor for plant growth and productivity in boreal forest

ecosystems, and thus, any continued enhancement of

forest NPP will require either increases in the availabil-

ity of resources or physiological adjustments that allow

increased uptake of these resources (Phillips et al.,

2011).

Many studies have reported an increased below-

ground C allocation and fine root production in trees

that are exposed to an elevated temperature (Majdi &

Ohrvik, 2004; Bai et al., 2010), indicating that the trees

are likely increasing their allocation to roots to explore

the soil for nutrients such as N (Johnson, 2006). How-

ever, since most limiting nutrients are locked up in soil

organic matter, merely increasing the amounts of roots

will be insufficient to sustain enhanced uptake rates.

Rather, trees will need to stimulate soil microbes to

release extracellular enzymes to access nutrients bound

up in soil organic matter (SOM) (Phillips, 2007; Drake

et al., 2011; Bengtson et al., 2012).Correspondence: Qing Liu, tel. 00 86 28 8522 9115,

fax 00 86 28 8522 2753, e-mail: [email protected]

2158 © 2013 Blackwell Publishing Ltd

Global Change Biology (2013) 19, 2158–2167, doi: 10.1111/gcb.12161

Microbial activity is generally limited by the avail-

ability of labile C in soil. Trees are known to stimulate

microbial activity and nutrient availability by releasing

root exudates (Phillips et al., 2009). Most exudates are

low molecular weight organic compounds that increase

nutrients due to their chelating properties or preferen-

tial use as substrates by soil microbes (Smith, 1976;

Phillips et al., 2012). In response to exudates, increases

in microbial activity and population growth may stimu-

late a microbial demand for nutrients, which can be

met by increasing the enzyme synthesis and the depo-

lymerization of N from SOM (Dijkstra et al., 2009). The

stimulation of SOM decomposition and accelerated N

cycling caused by inputs of labile C substrates has been

recently invoked as an important mechanism to explain

the long-term enhancement of forest productivity

under elevated CO2 (i.e., Rhizo-Accelerated Mineraliza-

tion and Priming or RAMP hypothesis; see Phillips

et al., 2012). In recent studies, we also have invoked dif-

ferences in root exudation to explain the changes in

rhizosphere effects and soil N transformations between

tree species under experimental warming (Yin et al.,

2012a,b). Despite the perceived importance of root exu-

dation to ecosystem function (Fransson & Johansson,

2010), there have been few measurements of the exuda-

tion rates from field-grown plants or mature trees

exposed to experimental warming. Thus, it is unknown

how this widely hypothesized but rarely quantified

process will influence SOM decomposition and N

cycling through the release of exudation under global

warming (Phillips et al., 2012).

Hence, in this study, we conducted an experiment

to examine plant root-microbe interactions in the soils

of Picea asperata plots under experimental warming

and varying N availability. The P. asperata species

was chosen because it is widely distributed and

important in the subalpine coniferous ecosystems in

western, Sichuan. Moreover, P. asperata primarily

functions as a keystone species in reforestation after

logging. We predicted that P. asperata species growing

under experimental warming would exude increased

C into the soils and that the enhanced rates of exuda-

tion would be associated with the increased enzyme

activity and stimulated soil N transformations. We

also predicted that the strength of these warming

effects would be reduced for the plots fertilized with

inorganic N. To test this hypothesis, we measured

differences in root exudation rates, soil N transforma-

tions and the associated enzyme activity in experi-

mental plots exposed to elevated temperature and/or

N fertilization. To our knowledge, this study is the

first study to investigate the interactive effects of

experimental warming and N fertilization on root

exudation and their impacts on soil N cycling.

Materials and methods

Experimental design

The experiment was conducted at the Maoxian Ecological

Station of the Chinese Academy of Sciences, Sichuan Province,

China (31°41′N, 103°53′E, 1820 m a.s.l.), where the mean

annual temperature, precipitation and evaporation are 8.9 °C,920 mm, and 796 mm, respectively. Our experiment followed

Wan et al. (2002) in using 165 9 15 cm infrared heaters (Kalgo

Electronics Inc., Bethlehem, PA, USA) to generate an artifi-

cially warmed environment. There were five pairs of 2 9 2 m

plots (a warmed plot and a control plot), and each 2 9 2 m

plot was divided into four 1 9 1 m subplots. The indigenous

soil of all subplots was replaced, to a depth of 50 cm, by

sieved topsoil from a coniferous forest. The soil was classified

as a mountain brown soil series (Chinese taxonomy). The soil

properties, determined in March 2007, were as follows: pH,

5.55; total N, 4.5 g kg�1; soil organic C, 78 g kg�1; and bulk

density, 0.89 g cm�3. The warmed plot was heated by an

infrared heater suspended 1.5 m above the middle of the

plots. The infrared heater had a radiation output of approxi-

mately 100 W m�2, and its warming effect on the soil temper-

ature was spatially uniform within the warmed plots. One

‘dummy’ heater with the same shape and size as the infrared

heater was suspended 1.5 m above each control plot to simu-

late the shading effects of the infrared heater in the warmed

plots.

Uniform 4 year old P. asperata seedlings from a local nurs-

ery were selected based on plant height and stem base diame-

ter. The average height and stem base diameter of the

P. asperata seedlings were 13.42 � 0.57 cm and 3.12 �0.45 mm, respectively. In March 2007, twenty healthy seed-

lings were planted randomly in separate subplots within each

plot. The seedlings that were grown in two of the diagonal

subplots of each plot were watered weekly with 200 ml of

2.7 mM ammonium nitrate solution (for a total equivalent to

25 g N m�2 a�1), and the seedlings in the other two subplots

were watered with the equivalent amount of water. Nitrogen

amounts were based on our previous studies (Yao & Liu,

2007; Zhao & Liu, 2009). The fertilizer was prevented from

moving between subplots by a 70 cm deep vertical polyvinyl

chloride board. Artificial warming and nitrogen addition were

conducted from April 2007 to the present. To disrupt the

potential effects of soil water on soil processes, the warmed

plots were watered as frequently as needed and were moni-

tored with a hand-held probe (IMKO, Ettlingen, Germany).

Moreover, all of the litter within the plots was removed once a

month to examine the pure effects of the tree species on the

soil processes via the roots and root exudation. The four treat-

ments in this study were as follows: (1) unwarmed unfertil-

ized (W0F0); (2) warmed unfertilized (W1F0); (3) unwarmed

fertilized (W0F1); and (4) warmed fertilized (W1F1).

Microclimate monitoring

Air temperature (at the height of 20 cm above the ground)

and relative humidity were measured using DS1923G

© 2013 Blackwell Publishing Ltd, Global Change Biology, 19, 2158–2167

ENHANCED EXUDATION STIMULATES N TRANSFORMATION 2159

temperature/humidity iButton data loggers, and soil tempera-

tures (5 cm depth) were measured using DS1921G Thermo-

chron iButton data loggers (DS1921G-F5, Maxim Integrated

Products; Dallas Semiconductor Inc., Sunnyvale, CA, USA) in

five pairs of plots at 60 min intervals during the experimental

period. The soil moisture was measured gravimetrically in soil

core samples (0–10 cm) that were collected once or twice a

month from April 2010 to December 2011. The soil samples

were dried at 105 °C for 12 h and were then weighed. The soil

moisture was expressed as a percentage of dry soil on a mass

basis.

Exudation measurements

Exudates were collected in May, late July and October of 2011

using a modified culture-based cuvette system developed

especially for root exudation collection in the field (Phillips

et al., 2008). The terminal fine roots (2 mm average diameter

with laterals) that were attached to the coniferous trees were

excavated from the topsoil (0–10 cm). The soil particles adher-

ing to the roots were carefully rinsed off with purified water

from a squirt bottle, and fine forceps were used to dislodge

SOM aggregates. The intact roots were placed into glass

cuvettes, filled with glass beads (c. 1 mm diameter) and sealed

with a special rubber septum. The rubber septum had a small

slit cut into it to accommodate the protruding root. The

cuvettes (including the controls with beads only) were cov-

ered in foil and reburied in the excavated area in the soil. After

a 2-day equilibration period, a fresh nutrient solution (0.5 mM

NH4NO3, 0.1 Mm KH2PO4, 0.2 Mm K2SO4, 0.2 mM MgSO4,

0.3 mM CaCl2) was flushed through each cuvette to remove

soluble C. After 24 h, ‘trap solutions’ containing exudates

were collected from each cuvette with an automatic electric

vacuum pump and were then placed on ice and filtered

through sterile 0.22 lm syringe filters within 2–5 h of collec-

tion. A detailed description of this method is available in

Phillips et al. (2008).

The exudates were collected randomly from two to three

roots in five subplots of each treatment. For each sampling

period, the exudates were collected over three consecutive

days and from different plants on each sampling date. All of

the roots were harvested following the final exudation collec-

tion of each root and were then scanned so that the morpho-

logical variables (i.e., fine root length, surface area, root tips,

etc.) could be quantified. All of the scanned images were visu-

ally inspected, calibrated using materials of known size, and

analyzed using WinRhizo software (Regents Instruments Inc.,

Qu�ebec, Canada).

The filtered trap solutions were analyzed for organic C on

a TOC analyzer (Multi N/C 2100; Analytic Jena, Jena,

Germany). The control cuvettes (beads only) were used to

account for C contamination resulting from nonexudates

sources. The exudation rates were calculated as the mass of C

(lg) flushed from each root system (minus the average C con-

centration in the control cuvettes) over the 24 h incubation

period. The exudation rates I (lg C g�1 root biomass h�1), II

(lg C cm�1 root length h�1), and III (lg C cm�2 root area

h�1) were calculated by dividing the total amount of C

flushed from the root system by the total fine root biomass,

the root length, and the root area, respectively, within each

cuvette.

Growth characteristics analysis

Five soil samples were taken from the topsoil (0–15 cm) with a

5-cm-diameter polyvinyl chloride core within each subplot.

The fine roots (� 2 mm) were carefully separated with fine

forceps, and the separated fine roots were carefully washed

and then analyzed with the WinRHIZO image analysis system

(Regent Instruments Inc., Sainte Foy, Qu�ebec, Canada), which

was used to measure the root length and the diameter of each

root. The roots were rinsed free of soil, and 0.5 g samples of

white, young roots were used immediately to assay fine root

activity (FRV) using the triphenyltetrazolium chloride (TTC)

method, as described by Basile et al. (2007). Ectomycorrhizal

infection was analyzed by counting the total number of

mycorrhizal tips per seedling and by calculating the extent of

the infection as the percentage of root tips that were mycorrhi-

zal (Dehlin et al., 2004). Moreover, five randomly selected

seedlings from each treatment were harvested in early August

2010 and were then divided into leaf, stem, and root compo-

nents. All of the plant parts were dried to a constant mass at

70 °C before measuring the dry weight. Total biomass, coarse

root biomass, fine root biomass, and the coarse root/fine root

mass ratio were calculated based on the measured data.

Soil enzyme activity and N transformation assay

The soil samples were collected from the topsoil (0–15 cm) in

early May, mid-July, and late September of 2011. The soils

were sampled within 1 week of an exudation measurement.

Three cores (3 cm in diameter, 15 cm deep) were randomly

taken from each subplot. The collected soil cores were mixed

to obtain one composite fresh sample for each subplot, and

the samples were immediately delivered on ice to the labora-

tory. Each composite sample was passed through a sieve

(2 mm diameter), and any visible living plant material was

manually removed from the sieved soil. The sieved soils were

kept in the refrigerator at 4 °C and were processed within

1 week for enzyme analysis.

We measured the activities of two extracellular enzymes

involved in the depolymerization of N from SOM. Urease is a

hydrolytic enzyme involved in the hydrolysis of urea-type

substrates. Given the chemistry of urea and its mass in the

soil, N released from SOM by urease is considered to be a

moderately fast cycling pool of N (Zhan et al., 2010). In con-

trast, phenol oxidase is a lignolytic enzyme involved in the

degradation of recalcitrant SOM, and an enzyme that is often

used as a sentinel of SOM decomposition (Sinsabaugh, 2010).

As lignin, tannins, and polyphenols may bind N, N released

from SOM by phenol oxidase is considered to be a relatively

slow cycling pool of N (Phillips et al., 2011).

Soil urease activity was measured as described previously

by Kandeler & Gerber (1988). Five grams of soil was placed in

a 50 mL Erlenmeyer flask, 1 mL of toluene was added to the

soil in the flask, and the contents were allowed to stand

for approximately 15 min until the toluene had completely


2160 H. YIN et al.

penetrated the soil. Then, a 20-mL potassium citrate-citric acid

buffer (pH 6.7) and 10 mL of a 10% urea solution were added

to the sample. The flasks were stoppered, shaken and then

incubated at 37 °C for 24 h. A control, in which 10 mL of dis-

tilled water was substituted for the urea, was examined simul-

taneously. After incubation, the contents of the flasks were

filtered. The amount of ammonia released by hydrolysis of the

urea was determined from the filtrate using the colorimetric

indophenol blue method. The unit of urease activity was

reported as mg of NH4+-N released per kg dry soil per 24 h.

Polyphenol oxidase was analyzed with pyrogallic acid as a

substrate. The mixture of 1 g soil and 10 mL of 1% pyrogallic

acid was incubated at 30 °C. A 4-mL disodium hydrogen

phosphate-citric acid buffer (pH 4.5) was added after 2 h incu-

bation, and purpurogallin was extracted with ether. The sam-

ple was then measured using a spectrophotometer set of a

wavelength of 430 nm. The polyphenol oxidase activity was

expressed as mg purpurogallin per g dry soil per 2 h (Zhou,

1987). All of the determinations of enzymatic activity were

performed in triplicates, and all of the values reported are the

averages of three trials performed on oven-dried soil (105 °C).The rates of net N mineralization and net nitrification in

May, July, and September were measured using the covered

core incubation method (Adams et al., 1989). We selected these

dates to coincide with a subset of the exudation sampling

dates. The incubations were performed using perforated PVC

tubes (15 cm in height and 6 cm in diameter). Parafilm was

used to cover the top of each tube to avoid leaching of nitrate.

This technique prevents the plant’s uptake of mineralized

nutrients but allows uptake by the microorganisms. The soil

samples were transported to the laboratory in a cool box and

were analyzed for ammonium and nitrate as the initial sample

to measure net mineralization and net nitrification rates. The

soil samples in the buried bags were retrieved after 30 days of

incubation and were analyzed as the final sample. The differ-

ence between the initial and final inorganic N concentrations

(NH4+-N and NO3

�-N) was used to calculate the net N miner-

alization rates. The difference between the initial and final

NO3�-N concentrations was used to calculate the net nitrifica-

tion rates.

The gross nitrification and denitrification rates were

measured using a Barometric Process Separation (BaPS)

instrument (UMS GmbH Inc., Munich, Germany) through lab-

oratory incubations, as described by Sun et al. (2009). Within

each subplot, three intact soil cores were collected using soil

containers with a diameter of 5.6 cm and a height of 4.1 cm.

The soil containers were transported to the laboratory in cool-

ers and were processed immediately. The BaPS instrument

was closed so that it was gas-tight, and the samples were incu-

bated for at least 24 h at 25.0 °C.

Statistical analyses

Analyses were performed with the software Statistical Pack-

age for the Social Sciences (SPSS) software, version 11.0 (SPSS

Inc., Chicago, IL, USA). All of the response variables were

averaged within each subplot, and the subplots were consid-

ered to be the experimental units. Before analysis, all of the

data were tested for the assumptions of ANOVA. If the data

were heterogeneous, they were ln-transformed before analy-

sis. A repeated measures ANOVA was used to assess the effects

of warming, N fertilization and their interactions on all of the

response variables. Because of our interests in the role of N in

mediating warming effects, a one-way ANOVA was also per-

formed to assess the effects of warming on root and soil vari-

ables at a given nutrient level and sampling date. We used

linear regression to examine the relationship between the exu-

dation rate and the extracellular enzyme activity and soil N

transformation. Given the limited number of soil samples,

data from all of the experimental plots were analyzed across

the sampling dates. The statistical tests were considered sig-

nificant at the P < 0.05 level.

Results

Warming effects of the infrared heaters

As expected, the infrared heaters caused warming

within the experimental plots. During the experimental

time period, the daily air temperature (at 20 cm above-

ground) and soil temperature (at 5 cm depth) within

the warmed plots were increased, on average, by 1.8 °Cand 3.6 °C, respectively, compared to the control plots

(Fig. 1a and b). The mean relative humidity of the air

was slightly lower in the warmed plots (85.1%) relative

to the control plots (94.5%) (Fig. 1c). Moreover, there

was no significant difference in the soil water content

between the control plots (25.7%) and the warmed plots

(24.6%) (Fig. 1d).

Warming and N fertilization effects on exudation

Our results showed that experimental warming had

significant effects on root exudation rates I, II, and III

(Table 1). Over the sampling dates, experimental

warming significantly increased root exudation rates I

(lg C g�1 root biomass h�1), II (lg C cm�1 root

length h�1), and III (lg C cm�2 root area h�1) in unfer-

tilized plots (Fig. 2), with an average exudation rate

increase of 78.1%, 68.6%, and 55.0%, respectively

(Fig. 2d). In contrast, experimental warming induced a

small but nonsignificant decrease in root exudation

rates I, II, and III, with an average decrease of 30.6%,

28.4%, and 24.2%, respectively (Fig. 2d). There were no

significant effects of N fertilization on root exudation

rates I, II, and III, and there were no warming 9 N

fertilization interactions (Table 1).

Growth traits response to treatments

Experimental warming significantly decreased the

coarse root/fine root mass ratio (C/F) for the P. asperata

seedlings, which may have resulted from relatively

more biomass partitioning to fine roots in response



to experimental warming in the unfertilized plots

(Fig. 3d). In contrast, experimental warming markedly

increased the fine root activity (FRV), fine root length

0

5

10

15

20

25

30

35D

aily

mea

n ai

r tem

pera

true Warmed plots

Control plotsTair

0

5

10

15

20

25

30

Dai

ly m

ean

soil

tem

pera

true Warmed plots

Control plotsTsoil

0

20

40

60

80

100

120

1-Mar

31-Mar

30-Apr

30-May

26-Jul

25-Aug

24-Sep

24-Oct

23-Nov

23-Dec

22-Jan

21-Feb

22-Mar

21-Apr

21-May

Date

Dai

ly m

ean

air r

elat

ive

hum

idity

Warmed plotsControl plots

RH

0

10

20

30

40

50

Apr MayJun Jul AugSep Oct NovDec FebMar Apr MayJun Jul AugSep Oct

Soil

wat

er c

onte

nt (%

) Warmed plotsControl plots

(a)

(b)

(c)

(d)

Fig. 1 Seasonal transitions and average differences in (a) daily

mean air temperature at 20 cm above ground, (b) daily mean soil

temperature (5 cm depth), (c) mean air relative humidity, and (d)

mean soil water content (0–10 cm) between the warmed plots (solid

line) and the control plots (dotted line). The lower gray lines (symbol

for △) in a, b, and c represent the daily mean differences in air tem-

perature, soil temperature, and air relative humidity, respectively,

between the warmed plots and the control plots. The scales of the

x-axis are 30-day intervals (a–c) from 1March 2010 to 25 May 2011.

Table

1Resultsoftherepeatedmeasu

res

ANOVAsh

owingthePvalues

fortheresp

onsesofrootexudationratesI(lgC

g�1rootbiomassh�1),II(lgC

cm�1rootlength

h�1),

andIII(lgC

cm�2rootarea

h�1),net

mineralization,net

nitrification,gross

nitrification,den

itrification,ureasean

dpolyphen

oloxidaseto

experim

entalwarming(W

),N

fertil-

ization(F),an

dsamplingdates

(D).Pvalues

less

than

0.05

arein

bold

Factor

ExudationI

ExudationII

ExudationIII

Net

mineralization

Net

nitrification

Gross

nitrification

Den

itrification

Urease

Polyphen

ol

oxidase

D0.001

<0.001

0.035

0.21

40.007

0.20

70.028

0.54

50.07

8

D9

W0.025

0.012

0.08

60.71

40.025

0.36

40.042

0.12

60.27

1

D9

F0.06

80.007

0.043

0.14

80.21

90.07

50.76

30.23

10.26

5

D9

W9

TS

0.21

50.19

20.32

50.36

20.39

80.10

80.06

70.47

80.27

9

W0.029

0.023

0.039

0.025

0.048

0.037

0.005

0.025

0.005

F0.05

20.05

70.06

80.96

30.039

0.12

30.24

90.034

<0.001

W9

F0.07

40.21

60.05

90.82

60.75

30.48

20.93

60.004

0.021


2162 H. YIN et al.

(FRL), fine root biomass, ectomycorrhizal infection

(EMI), and total biomass in the unfertilized plots, with

average increases of 19.8%, 66.0%, 62.1%, 56.7.0%, and

26.4% in FRV, FRL, fine root biomass, EMI and total

biomass, respectively (Fig. 3). In the fertilized plots,

however, experimental warming had no significant

effects on any root variable (Fig. 3). Moreover, N fertil-

ization significantly increased the total biomass of the

P. asperata seedlings in the unwarmed plots but not in

the warmed plots (Fig. 3f).

Soil N transformation response to treatments

Consistent with the response of root exudation, experi-

mental warming significantly increased the rates of net

mineralization, net nitrification, gross nitrification, and

denitrification on all of the sampling dates in the unfer-

tilized plots, with the exception of May, during which

there were no significant differences between the treat-

ments for gross nitrification rates (P = 0.654; Fig. 4).

Moreover, the net mineralization rates were positively

correlated with root exudation rate II (lg C cm�1 root

length h�1) (R2 = 0.699; Fig. 6a). In contrast, there were

no significant warming effects on any response vari-

able of N transformations in the fertilized plots among

the sampling dates (Fig. 4). The net nitrification and

denitrification rates significantly varied among three

sampling dates (P = 0.007 and 0.028, respectively;

Table 1).

Soil enzyme activity response to treatments

Warming and N fertilization had significant effects on

the extracellular activities of the two enzymes (Table 1;

P < 0.05). The soil enzyme activities in the unfertilized

plots responded more strongly to experimental warm-

ing compared with the enzyme activities in the fertil-

ized plots. Over the sampling dates, warming markedly

increased polyphenol oxidase activity by 38% in the

unfertilized plots, but it was increased by only 11.2% in

the fertilized plots (Fig. 5b). In contrast, N fertilization

significantly reduced the polyphenol oxidase activity

by 32.5% in May and by 36.4% in September. In addi-

tion, the urease activity was strongly correlated with

the root exudation rate II (lg C cm�1 root length h�1)

(R2 = 0.786; Fig. 6b).

Unlike the warming effects on polyphenol oxidase

activity, the warming effects on urease activity only

occurred in May, which significantly increased activi-

ties by 34.1% in the unfertilized soils, whereas there

were no considerable warming effects on the urease

activity in the fertilized plots in May or in the unfertil-

ized or the fertilized plots in September. Similarly,

N fertilization also significantly reduced the urease

0

400

800

1200

1600

2000

Exu

datio

n ra

te (µ

g C

–1g

root

bio

mas

s h–1

)

W0F0 W1F0W0F1 W1F1

(a) Exudation rates I

*

*

*

0

0.3

0.6

0.9

1.2

1.5

Exu

datio

n ra

te (µ

g C

cm

–1 r

oot l

engt

h h–1

)

(b) Exudation rates II

**

**

0

3

6

9

12

15

18

May Jul Oct

Exu

datio

n ra

te (µ

g C

cm

–2 r

oot a

rea

h–1)

(c) Exudation rates III

**

*

–60

–40

–20

0

20

40

60

80

100

War

min

g ef

fect

on

exud

atio

n (%

)

Control plotFertilized plot

**

**

*

Exudation rate I Exudation rate II Exudation rate III

(d)

Fig. 2 Effects of warming and N fertilization on exudation rates

I (lg C g�1 root biomass h�1; a), II (lg C cm�1 root length h�1;

b), and III (lg C cm�2 root area h�1; c) in the Picea asperata

seedlings. In (d), values represent the mean relative warming

effects on exudation rates I, II, and III from three sampling

dates. The white bars refer to the unfertilized subplots, and the

striped bars represent the N-fertilized subplots. For a–d, error

bars are � SD of the mean (n = 5), and significant differences

between the control plots and the warmed plots at a given nutri-

ent level (unfertilized or fertilized) are noted by asterisks

(**P < 0.01, * P < 0.05).



0

40

80

120

160

FRV

(µg.

g–1 F

W h

–1) Warmed plot

Control plot

(a)*

0

15

30

45

60

Roo

t len

gth

(cm

)

(b)*

0

4

8

12

16

20

Non-fertilized plots Fertilized plots

Fine

root

bio

mas

s (g)

(c)*

0

15

30

45

60

Myc

orrh

izal

infe

ctio

n (%

) (e)

*

0

40

80

120

160

200

240

Non-fertilized plots Fertilized plots

Tota

l bio

mas

s (g)

(f) *

0.0

0.8

1.6

2.4

3.2

4.0

Coa

rse/

fine

root

mas

s rat

io (d)*

Fig. 3 Effects of warming and N fertilization on fine root activity (FRV, a), fine root length (FRL, b), fine root biomass (FRB, c), coarse

root/fine root mass ratio (C/F, d), ectomycorrhizal infection (EMI, e), and total biomass (f) of the Picea asperata seedlings. Vertical bars

are means � SD, with a sample size of n = 5. Asterisks indicate significant differences (P < 0.05) between the control plots and the

warmed plots at a given nutrient level (unfertilized or fertilized).

0.0

0.2

0.4

0.6

0.8

May July September

Net

nitr

ifica

tion

(mg

N k

g–1 d

ay–1

)

*

* *

(b)

0.0

0.2

0.4

0.6

0.8

Net

min

eral

izat

ion

(mg

N k

g–1 d

ay–1

)

W0F0 W1F0W0F1 W1F1

*

(a)

*

*

0

2

4

6

8

10

12

Gro

ss n

itrifi

catio

n (m

g N

kg–1

day

–1)

**

(c)

0

1

2

3

4

5

6

7

May July September

Den

itrifi

catio

n (m

g N

kg–1

day

–1)

**

*(d)

Fig. 4 Effects of warming and N fertilization on rates of net mineralization (a), net nitrification (b), gross nitrification (c), and denitrifi-

cation (d) in the Picea asperata plots. Vertical bars are means � SD, with a sample size of n = 4. Asterisks indicate significant differences

(P < 0.05) between the control plots and the warmed plots at a given nutrient level (unfertilized or fertilized).


2164 H. YIN et al.

activity over the two sampling dates (Fig. 5b; Table 1).

In addition, significant combination effects of warming

and N fertilization were observed in the polyphenol

oxidase and urease activities in the soil (Table 1).

Discussion

The degree to which root exudation influences nutrient

cycling in forest soils is poorly understood because

associated ecological processes mediated by roots are

believed to be temporally and spatially heterogeneous

due to biotic (e.g., plant growth, litter input, tree age,

and belowground C flux) and abiotic factors (e.g., soil

moisture, fertility, pH, and nutrient availability) (Bader

& Cheng, 2007). Therefore, the influences of tree species

on soil processes and functions may be masked by the

pedology of the site, edaphic and environmental vari-

ability, and management effects. In this study, all of the

experimental seedlings were grown in a common soil

with similar field management, and the litter within the

subplots was removed periodically to disrupt the

effects of litter input on the soils. The differences in root

exudation and associated soil processes between treat-

ments were therefore assumed to reflect the potential

effects of a plant’s intrinsic physiological adjustment in

response to different environmental changes.

Warming and N effects on root exudation

Recent recognition of the importance of plant root–microbial–soil interactions has highlighted the need for

more information on the mechanisms by which trees

allocate C and cycle nutrients under environmental

change, and these interactions have major conse-

quences for the functioning of terrestrial ecosystems in

response to global climate change. There have been sev-

eral reports of CO2-induced changes in the root exuda-

tion rates of trees (Johansson et al., 2009; Phillips et al.,

2009; Fransson & Johansson, 2010), but there have been

few reports of the response of root exudation to experi-

mental warming in trees. In this study, our results indi-

cated that the exudation rates from P. asperata seedlings

are significantly increased by experimental warming

but such effects strongly depend on N availability

(Fig. 2). The belowground C allocation and root mor-

phological traits are thought to be the two primary

aspects controlling root exudates (Badri & Vivanco,

2009), and based on ancillary data, several possible

underlying mechanisms may explain the stimulatory

effects of experimental warming on exudation.

It is possible that greater root exudation in the low-N

soils resulted from the warming-induced changes in

root morphological traits. In our study, the root length

of the P. asperata seedlings was significantly enhanced

0.0

0.6

1.2

1.8

2.4

3.0Po

lyph

enol

oxi

dase

act

ivity

(mg

purp

urig

auin

g–1

dry

soil

2 h–1

)

W0F0 W1F0

W0F1 W1F1

(a)

**

0.0

0.6

1.2

1.8

2.4

3.0

May September

Ure

ase

activ

ity (m

g N

H4+ –

N k

g–1so

il da

y–1) (b)

*

Fig. 5 Effects of warming and N fertilization on soil polyphenol

oxidase activity (mg purpurogallin g�1 dry soil 2 h�1; a) and

urease activity (mg NH4+-N kg�1 soil d�1; b) in the Picea aspera-

ta plots. Vertical are means � SD, with a sample size of n = 4.

Asterisks indicate significant differences (P < 0.05) between the

control plots and the warmed plots at a given nutrient level

(unfertilized or fertilized).

y = 1.4592x + 0.5552R2 = 0.7896

0.0

0.5

1.0

1.5

2.0

2.5

0 0.2 0.4 0.6 0.8 1

Ure

ase

activ

ity (m

g N

H4+ -N

kg–1

soil

d–1)

Exudation rate (μg C cm–1 root length h–1)

y = 0.4626x + 0.1302R2 = 0.6994

0.0

0.2

0.4

0.6

0.8

Net

min

eral

izat

ion

rate

(mg

N k

g–1 d

–1)

(b)

(a)

Fig. 6 Relationship between the root exudation rate II

(lg C cm�1 root length h�1) and the net mineralization rate (a)

and soil extracellular enzyme activity (b) across all of the treat-

ments and sampling dates.



by warming in the unfertilized plots (Fig. 3b). A host of

studies have demonstrated that plant root length is pos-

itively correlated with root-released C (Xu & Juma,

1994; Darwent, 2003). Additionally, the FRV and ecto-

mycorrhizal infection of the P. asperata seedlings grown

in the unfertilized soils was remarkably higher in the

warmed plots relative to the control plots (Fig. 3a and

e). As a result, the capacity for enzyme synthesis, the

nutrient uptake mechanisms and the respiration rate of

the roots can differ for those trees grown under warm-

ing conditions, and these differences can strongly influ-

ence the quantity and chemical quality of root

exudation (Drake et al., 2011; Phillips et al., 2012).

In addition to altered root morphological traits, the

increased root exudation at the elevated temperature

might be associated with warming-induced changes in

belowground C allocation. An important compensatory

adjustment by plants exposed to environmental

changes is the altered allocation patterns of C to below-

ground tissues (Reich et al., 2006). Numerous growth

chamber experiments and field experiments have

reported an increased belowground production of C in

trees that were exposed to an elevated temperature

(Yin & Liu, 2008; Hollister & Flaherty, 2010). In this

study, our results indicated that the C/F ratio of the

P. asperata seedlings was significantly decreased by

experimental warming, resulting in increased C parti-

tioning to the fine roots in response to experimental

warming, presumably so that the roots could forage for

growth-limiting nutrients (Fig. 3c and d). Collectively,

these alterations will have profound impacts on the

quantity and chemical quality of root exudates and C

substrate inputs into the soils. However, the degree to

which such factors mediate root exudates of tree spe-

cies and feedbacks to soil ecological processes is

unknown. Further examination of root exudation in

response to environmental changes, with more detailed

characterization of root morphological and physiologi-

cal traits and belowground C allocation would be a

worthwhile focus of future studies.

Ecological consequences of enhanced exudation

Understanding the mechanism by which potential

changes in root-derived C affect the microbial regula-

tion of soil N cycling and nutrient availability under

experimental warming is critical for predicting biotic

feedbacks to climate change. In the present study, our

results indicated that increases in the flux of labile C

from the roots to the soil under experimental warming

conditions stimulated the rates of soil N transformation

in the unfertilized plots (Fig. 4), a mechanism that may

contribute to continuous stimulation of plant growth or

forest productivity under global warming. Increases in

the inputs of root-derived C can significantly stimulate

microbial activity and SOM decomposition via rhizo-

sphere priming effect (Dijkstra et al., 2009). Here, we

show that the increased labile C efflux from the

warmed trees stimulated the soil transformation rates

and the activity of two extracellular enzymes involved

in the breakdown of organic N in the unfertilized plots.

Importantly, the soil urease activity (an indicator of

moderately fast N turnover) and the net mineralization

rates were positively correlated with the measured

rates of exudation (Fig. 6). We interpret these results as

strong evidence of the influencing of the root-derived C

on the microbial regulation of soil N cycling, i.e., soil

heterotrophic microbes such as actinomycetes used

energy derived from the exudates to synthesize extra-

cellular enzymes to release N from SOM (Bengtson

et al., 2012; Phillips et al., 2012). This was not the case in

the N-fertilized plots, in which the root exudation, the

soil extracellular enzymes and the soil N transforma-

tion did not respond strongly to the experimental

warming. A possible explanation is that the soil

microbes in the high-N soils use C-rich exudates for

growth rather than for the production of enzymes to

acquire N (Drake et al., 2011). This dramatic contrast

between the fertilized and the unfertilized treatments

provides evidence that enhanced exudation is a mecha-

nism that trees employ to increase the soil N transfor-

mation and nutrient availability (Phillips et al., 2011).

It must be noted, however, that other physiological

adjustments by trees exposed to experimental warming,

such as fungal rhizomorph production and the alloca-

tion of C to ectomycorrhizal fungi (EMF), can also stim-

ulate soil N cycling. Although the available data on

EMF growth was limited in this study, our preliminary

experiments indicated that the EMF infection of the

P. asperata seedlings in the unfertilized plots was signif-

icantly increased by experimental warming (Fig. 3d).

EMF have broad enzymatic capabilities, decompose

labile and recalcitrant components of soil organic mat-

ter, access organic sources of N and transfer large

amounts of N to host plants (Hobbie & Hobbie, 2006;

H€ogberg & Read, 2006). Such changes would presum-

ably accelerate the N release from SOM pools. How-

ever, the degree to which EMF mediates the exudation

rates and priming effects in tree species exposed to

experimental warming warrants further study.

In conclusion, this study demonstrates that the

increase in the release of root exudation from trees

under experimental warming is an important physio-

logical adjustment that stimulates N cycling and nutri-

ent availability in low fertility soil. Although we fully

recognize the obvious limitations of our experimental

systems because all of the data came from small plants

growing in disturbed soils, our results are robust in


2166 H. YIN et al.

terms of the direction of treatment effects. Thus, our

results provide evidence that the degree to which trees

sequester C under global warming may depend on the

magnitude and ecological consequences of changes in

C released to the soil via root exudation. Accordingly,

the underlying mechanisms by which plant root-

microbe interactions influence soil organic matter

decomposition and N cycling should be incorporated

into climate-carbon cycle models to determine reliable

estimates of long-term C storage in forests.

Acknowledgments

We thank Jinsong Chen for assisting with the statistics, and YanZou and Bing Xia for their technical assistance in the laboratory.We also thank the staff in the Maoxian Mountain Ecosystem ofCERN Research Station for their kind help with field investiga-tions. This study was supported jointly by the National NaturalScience Foundation of China (No. 31270552), the strategic Prior-ity Research Program of the Chinese Academy of Sciences(No. XDA01050303) and the National Key Technology R & DProgram (No. 2011BAC09B04).

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