The effects of elevated CO2 on root respiration rates oftwo Mojave Desert shrubs

N A O M I M . C L A R K *1 , M A R T H A E . A P P L E w and R O B E R T S . N O WA K *

*Department of Natural Resources and Environmental Sciences, University of Nevada, Reno, Reno, NV 89557, USA, wDepartment

of Biological Sciences, Montana Tech of the University of Montana, Butte, MT 59701, USA

Abstract

Although desert ecosystems are predicted to be the most responsive to elevated CO2, lownutrient availability may limit increases in productivity and cause plants in deserts to allocatemore resources to root biomass or activity for increased nutrient acquisition. We measuredroot respiration of two Mojave Desert shrubs, Ambrosia dumosa and Larrea tridentata, grownunder ambient (� 375 ppm) and elevated (� 517 ppm) CO2 concentrations at the NevadaDesert FACE Facility (NDFF) over five growing seasons. In addition, we grew L. tridentataseedlings in a greenhouse with similar CO2 treatments to determine responses of primary andlateral roots to an increase in CO2. In both field and greenhouse studies, root respiration wasnot significantly affected by elevated CO2. However, respiration of A. dumosa roots o1 monthold was significantly greater than respiration of A. dumosa roots between 1 and 4 months old.For both shrub species, respiration rates of very fine (o1.0 mm diameter) roots weresignificantly greater than those of fine (1–2 mm diameter) roots, and root respiration decreasedas soil water decreased. Because specific root length was not significantly affected by CO2 andbecause field minirhizotron measurements of root production were not significantly different,we infer that root growth at the NDFF has not increased with elevated CO2. Furthermore, otherstudies at the NDFF have shown increased nutrient availability under elevated CO2, whichreduces the need for roots to increase scavenging for nutrients. Thus, we conclude thatA. dumosa and L. tridentata root systems have not increased in size or activity, and increasedshoot production observed under elevated CO2 for these species does not appear to beconstrained by the plant’s root growth or activity.

Keywords: Ambrosia dumosa, atmospheric CO2, Larrea tridentata, Mojave Desert, root respiration,

specific root length

Received 18 May 2009; revised version received 28 August 2009 and accepted 7 September 2009

Introduction

The recent rise in atmospheric CO2 concentrations

resulting from fossil fuel burning and deforestation is

the most widespread, documented, and observed ex-

ample of human influence on the earth’s atmosphere

(Vitousek et al., 1997). The magnitude of this effect is

expected to be greatest in desert ecosystems, where

elevated atmospheric CO2 concentrations are thought

to increase plant water use efficiency (Melillo et al.,

1993). Indeed, deserts have among the greatest in-

creases in aboveground productivity in response to

CO2 enrichment (Nowak et al., 2004) with the greatest

responses occurring during wet years (Smith et al., 2000;

Housman et al., 2006).

At the Nevada Desert FACE Facility (NDFF), above-

ground productivity and photosynthesis have increased

with elevated CO2 in the Mojave Desert (Smith et al.,

2000; Housman et al., 2006). With increases in carbon

assimilation under elevated CO2 concentrations, below-

ground resources are hypothesized to restrict plant

growth, especially in the water and nutrient limited

Mojave Desert (Titus et al., 2002). Therefore, alterations

in aboveground physiology likely influence the alloca-

tion of recent photosynthate to roots. For example,

plants may increase carbon allocation to root systems

for nutrient or water uptake, mycorrhizal symbioses or

further soil exploration. These investments can lead to

greater carbon losses via root turnover, exudation, and

respiration (Hungate et al., 1997). Because rhizosphere

processes are strongly related to nutrient uptake and

cycling, these activities must be adequately understood

1Current Address: Department of Plant Sciences, University of

California at Davis, Davis, CA 95616, USA.

Correspondence: Robert S. Nowak, tel. 1 775 784 1656, fax 1 775

784 4789, e-mail nowak@cabnr.unr.edu

Global Change Biology (2010) 16, 1566–1575, doi: 10.1111/j.1365-2486.2009.02075.x

1566 r 2009 Blackwell Publishing Ltd

to better predict ecosystem changes such as plant

growth and carbon storage with elevated CO2.

Although both root structure and function influence

rhizosphere processes, more studies have focused on

root biomass responses to elevated CO2 than on root

physiological processes. Plant roots typically display a

pronounced increase in growth under enhanced CO2

conditions (Rogers et al., 1994; Hodge & Millard, 1998;

George et al., 2003), but this response depends on species

(Gorissen, 1996; Cotrufo & Gorissen, 1997). Larrea triden-

tata, a dominant shrub in the Mojave Desert and other hot

deserts of western North America, has not exhibited

consistent changes in belowground biomass in response

to elevated CO2 in controlled growth chamber and green-

house experiments. Obrist & Arnone (2003) found ele-

vated CO2 increased the total biomass of L. tridentata,

including the roots, while others have reported no in-

crease in belowground biomass (BassiriRad et al., 1997;

Huxman et al., 1999). Unfortunately, in most root biomass

studies, distinctions are not generally made on the rela-

tive contributions of active and non-active root to bio-

mass values, and these results can be misleading. For

example, root tips constitute a small portion of root mass,

but are the primary site of many physiological processes

(Norby, 1994; Cotrufo & Gorissen, 1997). Therefore,

changes in belowground biomass must be interpreted

cautiously, and root physiology should be further re-

searched to gain a clearer understanding of how below-

ground processes respond to elevated CO2.

Previous studies investigating the influences of CO2 on

root respiration in various plant species range widely in

their results. Increases (Janssens et al., 1998; Edwards &

Norby, 1999), no change (Tjoelker et al., 1999; Yoder et al.,

2000; George et al., 2003), and decreases (BassiriRad et al.,

1996) have all been reported. By examining the effects of

elevated temperature and CO2 concentrations on sugar

and red maples, Edwards & Norby (1999) found that the

respective rhizospheres of these species respond differ-

ently to these particular environmental variables. Because

average study site temperatures and species composition

differ among experiments, these factors may be respon-

sible for varying responses of root respiration to elevated

CO2. The lack of a universal response to elevated CO2

highlights the need for experiments testing CO2 effects in

specific systems.

At the NDFF, soil respiration rates are significantly

greater with elevated CO2 (A. de Soyza et al., unpub-

lished data). Because we have not observed increased

root growth (Phillips et al., 2006) or mycorrhizae (Clark

et al., 2009) under elevated CO2 in the Mojave Desert at

the NDFF, we hypothesized that plants at the NDFF

increased carbon allocation to root activity rather than

biomass with elevated CO2, thereby contributing to

greater soil respiration. Our objective was to determine

the effects of elevated CO2 on the root respiration rates

of Ambrosia dumosa and L. tridentata, two dominant

shrubs in the Mojave Desert, during both greenhouse

and field experiments. In this study, we examined fine

roots, which are the sites for uptake of most nutrients

and water and thus are most likely to respond to

increased carbon input.

Materials and methods

Greenhouse experiment

CO2 treatment. We grew L. tridentata seedlings under

two different CO2 concentrations (360 and 550 ppm) in

the Frits Went Greenhouse at the University of Nevada,

Reno. We did not include A. dumosa in this experiment

due to the difficulty in cultivating this species. The

greenhouse has two climate-controlled bays and is fitted

with a CO2 injection and scrub system allowing the two

bays to be maintained at different CO2 concentrations.

Night : day temperatures were set to 20 : 37 1C to mimic

those of a Mojave Desert spring and summer. To avoid

confounding bay effects, CO2 treatments and their

corresponding plants were transferred between bays

throughout the experiment.

L. tridentata seeds were placed in cheesecloth slings

under a dripping water faucet for 2 days to leach away

germination inhibitors. The seeds germinated under their

respective CO2 treatment in water-saturated, sterilized

sand in transparent, domed trays that maintained high

humidity. After 4 weeks, seedlings were either harvested

or transplanted to 5.5 L PVC pots filled with sand. Once

transplanted in September 2001, seedlings were given

100 mL H2O every 4 days. In addition, pots were

supplemented with 50 mL of 1/4 strength Hoagland’s

solution once per week until plants were harvested after

10 months.

Harvests. We harvested 16 seedlings per CO2 treatment

at 4 weeks (Fall 2001) and harvested the remainder of

the seedlings (20 plants per treatment) at 10 months

(Summer 2002). We measured root respiration as a func-

tion of oxygen consumption with a Clark-style oxygen

electrode (Hansatech Oxytherm, Hansatech Instru-

ments Ltd, Norfolk, UK). Immediately after harvest,

excised roots were placed in an aqueous physiological

buffer (10 mM MES, 1 mM CaSO4), and then root

respiration was measured for 10–15 min at 20 1C. We

measured the length of excised roots, and following

respiration measurements, we determined dry root

mass to calculate specific root length (SRL; m root g�1

dry mass). Although root diameter classes were defined

as fine (1–2 mm diameter) and very fine (o1 mm

diameter), almost all primary and lateral roots were in

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the very fine root diameter class for these very young

seedlings. Finally, we measured total dry mass of

aboveground and belowground biomass, and then com-

puted the root mass fraction (i.e. the proportion of total

biomass that was root mass). Roots were then combusted

and analyzed with a Perkin Elmer Instruments (Shelton

CT, USA) Model 2400 Series II CHNS/O Analyzer for

carbon and nitrogen content.

Statistical analyses. For 4-week-old seedlings, plant mass

and root mass fraction were analyzed using a one-way

ANOVA (PROC GLIMMIX, SAS 9.1) with CO2 concentra-

tion as the main effect. Root mass, respiration, and SRL

were analyzed with a two-way split-plot ANOVA (root

type�CO2) to determine CO2 effects on primary and

lateral roots. The individual plant was treated as the

experimental unit with root type as a split-plot treat-

ment. Data were transformed to meet ANOVA assump-

tions using the Box-Cox family of transformations

((yl�1)/l), with l selected using PROC TRANSREG

(SAS 9.1).

For 10-month-old seedlings, root and shoot biomass,

root mass fraction, very fine root respiration, and very

fine root SRL were analyzed with ANOVA (PROC

GLIMMIX, SAS 9.1). The main effect was CO2 concen-

tration. Again, data were transformed to meet ANOVA

assumptions using the Box-Cox family of equations

with the best l determined by PROC TRANSREG (SAS

9.1). We conducted similar analyses on carbon, nitrogen,

and C : N ratios of very fine and fine roots. All percent

carbon and nitrogen data were transformed using

arcsine square root, and C : N ratios were transformed

using log10.

Field experiment

Site description. The NDFF, 970 m asl on Frenchman Flat

at the Nevada Test Site, Nevada, USA, is a research

facility designed to study responses of an undisturbed

Mojave Desert ecosystem to increasing atmospheric

CO2 using free air CO2 enrichment (FACE) technology

(Jordan et al., 1999). Temperature extremes range from

48 1C to �19 1C, and precipitation is sporadic, averaging

� 140 mm annually. At the NDFF, which began

operation in April 1997 and ran continuously until

July 2007, six 23 m diameter study plots were used in

our study: three elevated CO2 FACE plots (CO2 set

point of � 550 ppm; average CO2 concentration

across all dates during 2001–2006 was � 517 ppm)

and three control rings fumigated at ambient CO2

(� 375 ppm) (http://www.unlv.edu/Climate_Change_

Research/). The soils are well drained Aridisols on an

alluvial fan with low soil carbon (0.18–1.8%) and

nitrogen (0.01–0.08%) concentrations (Jordan et al.,

1999). Vegetation is codominated by L. tridentata, an

evergreen shrub that grows over 1 m in height, and

A. dumosa, a smaller, drought-deciduous shrub.

Root observation boxes. We accessed roots of L. tridentata

and A. dumosa via small root observation boxes measur-

ing 60 cm� 45 cm� 30 cm with removable 50 cm� 20 cm

Plexiglas front windows that were perpendicular to but

located below the soil surface. Root boxes were installed

in 2000 by digging a square pit into the soil near the base

of two plants per species per plot and then placing a root

box in each soil pit (for a total of 24 boxes). The square pit

was slightly larger in cross-sectional area than the root

box to allow the root box to easily fit into the soil pit.

When inserting the box into the pit, the Plexiglas front

window was placed against undisturbed soil near the

base of the target plant, and then native soil (sieved to

remove gravel and rocks) was used to backfill any gaps

between the box and existing soil. Boxes were tightly

fitted with a lid that was constructed from a metal

frame and doubled layer of dense carpeting that was

sandwiched between metal hardware cloth. The carpet

helped minimize disturbance to the plant’s rooting

environment by: (1) intercepting sunlight such that the

interior of the root boxes stayed dark and roots were not

unnaturally affected by light; (2) providing thermal

insulation to help maintain natural soil temperatures

for roots growing near the root box; (3) absorbing rain

so that run-off from the box lid to the surrounding soil

did not occur, which would artificially increase soil

water content for surrounding roots; and (4) allowing

some water from large rain events to drip through the

interior of the box to deeper soils so that deeper soil

under the root box was not artificially dry. Styrofoam

also was placed against the Plexiglas window when not

in use to help maintain ambient soil temperature and a

dark environment. Boxes were accessed from moveable

platforms to avoid soil disturbance. Baseline tracings of

existing roots were made on sheets of transparent mylar

fitted to the Plexiglas windows each November before

the start of the growing season. Roots were monitored

and new roots were traced in date-specific colors at 2–4

week intervals until major root growth ceased in May or

June. The age of an individual root at the date of harvest

was determined from these tracings.

Respiration measurements. We measured individual

2–4 cm long excised end-of-root segments of known

age and diameter class. Excised roots were placed in

an aqueous physiological buffer (10 mM MES, 1 mM

CaSO4) in order to equilibrate for the 10 min

immediately after harvest. Respiration was measured

with a Hansatech Oxytherm Clark-style oxygen

electrode for 10–15 min at 20 1C by placing one root

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segment in oxygenated buffer in the cuvette of the

Oxytherm. We dried root samples for 48 h at 60 1C to

determine dry mass. Roots of two diameter classes

(very fine o1.0 mm diameter; fine 1–2 mm diameter)

were harvested in 2001, 2002, and 2003, but only very

fine roots were sampled in 2005 and 2006. Root age

varied from o1 month to � 4 months for roots

measured in 2001–2003, but only young roots

(o1 month) were sufficiently present in 2005 and

2006. Lengths and dry mass of roots were used to

calculate SRL (m root g�1 dry mass). Roots were

combusted in a Perkin Elmer Instruments Model 2400

Series II CHNS/O Analyzer to determine carbon and

nitrogen content.

Statistical analysis. Roots younger than 1 month were

categorized as ‘young’ for comparisons across years.

‘Old’ roots were defined as those roots older than

1 month. For each species, we conducted two

statistical analyses for root respiration and SRL. The

first analysis consisted only of data from 2001 to 2003

and included roots of different diameter and age classes

because fine roots and older roots were only abundant

enough for harvest in 2001, 2002, and 2003. Data for

2001–2003 were analyzed with a three-factor, split-plot

ANOVA using PROC GLIMMIX (SAS 9.1). The factors in

the analysis were CO2, diameter, and age. Age and

diameter were treated as split-plot treatments within

the main CO2 plots, and the ANOVA model included all

two- and three-way interaction terms. Data were

transformed to meet ANOVA assumptions using the

Box-Cox family of transformations with the best ldetermined by PROC TRANSREG (SAS 9.1). Owing to

the scarcity of fine roots, we were unable to consider

sampling date in this analysis, but pooled data across

years. Each plot was considered the unit of replication,

and the average respiration rate of roots from each root

box for very fine and fine roots between 2001 and 2003

was considered a subsample of each plot.

Because young, very fine roots were abundantly

available for harvest in 2005–2006, the second analysis

of root respiration and SRL included data from all

young, very fine roots across all years. We employed a

two-factor split-plot ANOVA (PROC GLIMMIX SAS 9.1)

for data analyses. We treated CO2 as the whole plot

factor and sampling date as the split-plot factor.

Individual root boxes (two per species per plot) were

considered to be subsamples. CO2 and sampling date

were the main effects, and the two-way ANOVA model

included the interaction term. Data were Box-Cox

transformed using the best l as determined by PROC

TRANSREG (SAS 9.1).

Results

Greenhouse experiment

Four-week-old seedlings. Although elevated CO2

significantly increased total plant biomass production

(P 5 0.010), it did not significantly affect root chara-

cteristics of 4-week-old L. tridentata seedlings (Table 1).

L. tridentata seedlings grown under elevated CO2 were

40% larger than those under ambient conditions.

Almost 60% of this increase in total biomass was due

to a significant increase in shoot biomass with elevated

CO2 (P 5 0.049). Although average primary and lateral

root biomass also increased with elevated CO2, these

differences were not significant (P 5 0.253). Root mass

fraction (the proportion of total biomass being root)

also did not respond significantly to elevated CO2

Table 1 Means and standard errors for parameters measured on 4-week-old Larrea tridentata seedlings grown in a greenhouse

under ambient and elevated CO2

Parameter

Ambient CO2 Elevated CO2

Mean (� SE) Mean (� SE)

Total plant biomass (mg) 6.717a (� 0.715) 9.634b (� 0.773)

Root mass fraction 0.240 (� 0.043) 0.358 (� 0.040)

Lateral root biomass (mg) 0.591 (� 0.195) 1.236 (� 0.322)

Primary root biomass (mg) 1.091 (� 0.202) 1.751 (� 0.266)

Shoot biomass (mg) 5.110a (� 0.536) 6.785b (� 0.685)

Lateral root respiration (mmol O2 min�1 m�1) �0.0303 (� 0.0161) �0.0171 (� 0.0038)

Primary root respiration (mmol O2 min�1 m�1) �0.0092 (� 0.0028) �0.0116 (� 0.0016)

Lateral root specific root length (m g�1) 59.83 (� 17.84) 72.85 (� 27.32)

Primary root specific root length (m g�1) 63.62 (� 13.01) 39.93 (� 4.69)

Means within a row followed by a different letter are significantly different.

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(P 5 0.080) but tended to increase with elevated CO2.

Lateral root respiration tended to be greater than

primary root respiration, but this difference was also

not significant (P 5 0.083). Neither CO2 (P 5 0.713) nor

its interaction with root type (P 5 0.484) significantly

affected root respiration. SRL was not significantly

affected by CO2 (P 5 0.332), root type (P 5 0.614), or

their interaction (P 5 0.714).

Ten-month-old seedlings. Root and shoot biomass as well

as root mass fractions of 10-month-old L. tridentata

seedlings were significantly affected by the CO2

treatments. Root biomass averaged 200% greater with

elevated CO2 (Po0.001; Table 2). Shoot biomass was

also greater with the elevated CO2 treatment (P 5 0.020),

although the relative increase (50%) was smaller than

that for root biomass. Consequently, root mass fractions

were significantly greater with elevated CO2 (P 5 0.012).

Very fine root carbon and nitrogen concentrations

(Table 2) were not significantly affected by CO2

(P 5 0.344 and 0.657, respectively), and consequently,

C : N ratios also were not influenced (P 5 0.606). Fine

root carbon (P 5 0.105) and nitrogen (P 5 0.681)

concentrations and C : N ratios (P 5 0.296) also were

not significantly affected by CO2 treatment.

Very fine root respiration was not significantly

affected by CO2 treatment (P 5 0.707; Table 2). Averaged

across CO2 treatments, L. tridentata seedlings’ root

respiration was �0.0140 � 0.0014mmol O2 m�1 min�1. In

addition, very fine root SRL of L. tridentata seedlings was

not significantly affected by CO2, (P 5 0.610). Averaged

across CO2 treatments, SRL of L. tridentata seedling very

fine roots was 159 � 17 m g�1.

Field experiment

Root respiration. Root respiration rates of adult A. dumosa

and L. tridentata growing in the field at the NDFF were

not significantly affected by CO2 treatment (Table 3,

Figs 1 and 2). CO2 interactions with age class and dia-

meter class were also not significant for both species

(Table 3A). For A. dumosa roots, age and age�diameter

were significant in the ANOVA. Averaged over both CO2

treatments and both root diameter classes, respiration

rates of young (o1-month-old) A. dumosa roots were

significantly greater than those of old (1–4 months old)

roots (Fig. 1). Although this ranking of means occurred

for both fine and very fine roots, mean comparisons

from the significant age�diameter interaction term did

not show significant differences between age classes

within each diameter class. However, for old A. dumosa

roots, fine roots had significantly lower respiration rates

than very fine roots. For L. tridentata, diameter was

the only significant effect in the ANOVA. Very fine

L. tridentata roots had greater respiration rates than

fine roots. Root respiration averaged across all CO2

treatments, ages, and diameters was �0.018 � 0.002

and �0.017 � 0.002mmol O2 min�1 m�1 for A. dumosa

and L. tridentata, respectively, on a unit length basis.

When expressed on a per mass basis, root respiration

rates averaged �25.3 � 5.7 and �20.5 � 7.5 nmol

O2 s�1 g�1 for A. dumosa and L. tridentata, respectively.

Although the overall main effect of CO2 was

not significant for very fine root respiration of both

A. dumosa and L. tridentata, rates significantly differed

among sampling dates and between CO2 treatments on

some sampling dates (Table 3B). For A. dumosa,

significant differences between CO2 treatments

occurred on only three of 15 sample dates (Fig. 2a).

On two of those dates (May 2003 and May 2005),

respiration of very fine roots for plants grown under

elevated CO2 was significantly greater than that under

ambient CO2, but respiration rates under ambient CO2

were greater on the other sample date (April 2002).

Significant differences between CO2 treatments for

L. tridentata only occurred in 2006 (Fig. 2b), and as for

A. dumosa, plants grown under elevated CO2 had

significantly greater root respiration for two sample

dates (February and April 2006) but respiration rates

Table 2 Means and standard errors for biomass fractions and

carbon and nitrogen contents of 10-month-old Larrea tridentata

seedlings in ambient and elevated CO2 treatments

Measurement

Ambient CO2 Elevated CO2

Mean (� SE) Mean (� SE)

Biomass

Root (g) 2.08a (� 0.23) 4.56b (� 0.54)

Shoot (g) 1.50a (� 0.15) 2.01b (� 0.15)

Root mass fraction 0.573a (� 0.026) 0.668b (� 0.025)

Carbon content

Very fine roots 28.5 (� 2.3) 31.5 (� 2.2)

Fine roots 31.1 (� 3.0) 37.3 (� 2.0)

Nitrogen content

Very fine roots 0.83 (� 0.07) 0.87 (� 0.07)

Fine roots 0.65 (� 0.08) 0.69 (� 0.06)

C : N ratio

Very fine roots 35.0 (� 1.7) 37.3 (� 2.6)

Fine roots 51.0 (� 3.1) 56.5 (� 4.0)

Root respiration

(mmol O2

min�1 m�1)

�0.0136 (� 0.0022) �0.0143 (� 0.0019)

Specific root length

(m g�1 root)

154 (� 28) 163 (� 22)

Means within a row followed by a different letter are signifi-

cantly different.

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under ambient CO2 were greater in May 2006. CO2

treatments were not significantly different for the

remaining 12 sample dates. Differences among sample

dates within each year across CO2 treatments were not

significantly different in 2001, 2002, 2003, and 2006 for

both shrub species (Fig. 2a and b). In 2005 (the most

intensively sampled year), maximum root respiration

for both species occurred in earlier months when soils

were near their maximum water content (Fig. 2c).

Percent soil water content declined towards the end of

the 2005 sampling season, and root respiration rates

also declined as soils became drier.

SRL. CO2 did not significantly affect the SRL of fine or

very fine roots of either species (Table 3). CO2

interactions with age and diameter also were not

Table 3 ANOVA tables for root respiration and specific root length of adult Ambrosia dumosa and Larrea tridentata for: (A) roots of

different size and age classes measured during 2001–2003 measured per unit mass; and (B) young, very fine roots measured during

all 5 years of sampling (2001, 2002, 2003, 2005, and 2006) measured per unit length

Effect

Root respiration Specific root length

Ambrosia dumosa Larrea tridentata Ambrosia dumosa Larrea tridentata

Num

df

Den

df

P

value

Num

df

Den

df

P

value

Num

df

Den

df

P

value

Num

df

Den

df

P

value

(A) All root size and age classes, but only 2001–2003 sample dates

CO2 1 4 0.924 1 4 0.617 1 4 0.714 1 4 0.372

Diameter 1 124 0.162 1 83 0.001 1 154 0.060 1 100 o0.001

CO2�diameter 1 124 0.756 1 83 0.346 1 154 0.472 1 100 0.572

Age 1 124 0.009 1 83 0.671 1 154 0.222 1 100 0.650

Age�diameter 1 124 0.052 1 83 0.659 1 154 0.016 1 100 0.600

CO2� age 1 124 0.677 1 83 0.258 1 154 0.612 1 100 0.379

Age�CO2�diameter 1 124 0.456 1 83 0.790 1 154 0.654 1 100 0.670

(B) All sample dates, but only young, very fine roots

CO2 1 4 0.760 1 4 0.161 1 4 0.896 1 4 0.180

Sample date 11 154 o0.001 12 173 o0.001 11 172 o0.001 12 184 o0.001

CO2� sample date 11 154 0.015 12 173 0.050 11 172 0.355 12 184 0.686

Bold P values are considered statistically significant.

Fig. 1 Respiration rates per unit dry mass of adult A. dumosa and L. tridentata roots grown under field conditions in ambient and

elevated CO2 plots at the NDFF. Untransformed means and standard error bars are shown for two root diameter classes (very fine

o1.0 mm; fine 1–2 mm) and for two age classes (young o1 month; old 1–4 months). Respiration rates are not significantly different

between CO2 treatments, but significant differences among diameter–age class combinations are shown by different lower case letters

below the bars. Data were averaged over all roots sampled during 2001, 2002, and 2003.

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significant (Table 3A). However, SRL differed

significantly among diameter classes for L. tridentata;

for A. dumosa, differences between diameter classes

were significant at P 5 0.06. SRL of very fine roots for

A. dumosa and L. tridentata (106 � 10 and 171 �28 m g�1, respectively) were 2.5� and 6.8� greater

than that of fine roots (42 � 27 and 25 � 37 m g�1,

respectively). In addition, the root age�diameter

interaction was significant for A. dumosa but not for

L. tridentata (Table 3A). For A. dumosa, older fine roots

(24 � 22 m g�1) had significantly lower SRL than older

very fine roots (105 � 10 m g�1), but differences in SRL

between newer fine (59 � 29 m g�1) and newer very fine

(108 � 10 m g�1) were not significant.

SRL of very fine roots was significantly different

among sampling dates for both species; however,

CO2� sampling date was not a significant interaction

(Table 3B). Unlike root respiration rates, SRL differences

among sampling dates do not appear to be related to

seasonal changes in soil moisture, and no sample dates

within the same year had significantly different SRLs.

Discussion

Elevated CO2

In both greenhouse and field experiments, elevated CO2

did not significantly affect root respiration rates of

either A. dumosa or L. tridentata (Tables 1–3; Figs 1 and

2), two dominant shrubs in the Mojave Desert of wes-

tern North America. Although the CO2� sample date

interaction was significant for young, very fine roots of

both species, CO2 effects were not significant on 80% of

the sample dates and not consistently greater for either

of the CO2 treatments. Elevated CO2 also has not

affected root respiration rates for three Mojave Desert

Fig. 2 Respiration rates per unit root length of adult (a) A. dumosa and (b) L. tridentata very fine roots grown in ambient and elevated

CO2 field plots at the NDFF across all sampling dates. Data are presented as untransformed means and standard errors. *Significant

difference between ambient and elevated CO2 treatments on a particular sample date. Sample dates in 2005 with the same letter were not

significantly different for root respiration within a species; sample dates within a year were not significantly different in all other years

sampled. (c) Percent soil water content at 15 cm depth across sampling dates in ambient and elevated CO2 treatments.

1572 N . M . C L A R K et al.

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grasses, Bromus madritdensis ssp. rubens, Achnatherum

hymenoides, and Pleuraphis rigida (Yoder et al., 2000).

Although similar results have been observed in other

ecosystems (Tjoelker et al., 1999; George et al., 2003),

contrary results have also been reported (BassiriRad

et al., 1996; Janssens et al., 1998; Edwards & Norby,

1999), making generalizations on how elevated CO2

affects root respiration difficult. Nitrogen contents of

A. dumosa and L. tridentata roots sampled from the

elevated CO2 plots at the NDFF are not significantly

different from those from ambient CO2 plots (R.S. Nowak

et al., unpublished data), and L. tridentata root nitrogen

also was not affected in our greenhouse experiment after

10 months of CO2 treatment (Table 2). These similar

nitrogen contents may help explain the similar root

respiration rates, which are highly correlated with nitro-

gen concentrations in root tissue across ecosystems

(Burton et al., 2002).

Even when conceptually scaled up to the collective

root systems of A. dumosa and L. tridentata in our field

study, our results indicate that root respiration rates of

the entire root system were not greater with elevated

CO2. Minirhizotron images at the NDFF do not

show significant changes in root length of A. dumosa

or L. tridentata with elevated CO2 treatment (Phillips

et al., 2006). Because we did not observe a change in SRL

of either species (Table 3), we can also infer that below-

ground fine root biomass for these two species is also

unaffected by the elevated CO2 treatment.

Contrary to the field results at the NDFF, where root

length of adult shrub communities treated with elevated

CO2 for up to 10 years were not significantly different

(Phillips et al., 2006; S. Ferguson et al., unpublished data),

L. tridentata root systems for 10-month-old greenhouse

seedlings were larger under elevated CO2 (Table 2).

However, the increase in root biomass after 10 months

may be short-lived and restricted to the early develop-

mental periods of seedlings. For example, Obrist &

Arnone (2003) noted that stimulation of root growth in

L. tridentata with elevated CO2 declined after 100 days

until their final harvest at 178 days. Greater root invest-

ments in early developmental stages with elevated CO2

may be an attempt to increase the likelihood of seedling

establishment. Indeed, at the NDFF, early survival of L.

tridentata seedlings was greater in the elevated CO2 plots

(Housman et al., 2003); however, this effect did not

continue with severe drought.

Compared with species found in other ecosystems,

our measured rates of root respiration for A. dumosa and

L. tridentata are slightly higher. Fine (1–2 mm diameter)

A. dumosa and L. tridentata roots respire at average rates

of �14 and �3 nmol O2 s�1 g�1, respectively (Fig. 1).

Unfortunately, no known studies have reported respira-

tion rates of desert shrubs for comparison with our

study. In other ecosystems, sugar maple roots of similar

diameter have respiration rates between �1 and

�10 nmol O2 s�1 g�1 (Pregitzer et al., 1998); our measure-

ments for A. dumosa are slightly higher than these for

sugar maple whereas L. tridentata falls within the range.

Very fine roots (o1 mm, although frequently o0.5 mm

diameter) of A. dumosa and L. tridentata under field

conditions respire at average rates of �36 and

�38 nmol O2 s�1 g�1, respectively; the average rate for

10-month-old L. tridentata under greenhouse conditions

is �37 nmol O2 s�1 g�1. Sugar maple roots o0.5 mm

diameter collected in Michigan respired at rates

between �5 and �20 nmol O2 s�1 g�1 (Pregitzer et al.,

1998), which are rates lower than those we measured for

two desert shrubs. Various gymnosperm and angios-

perm tree roots o1 mm diameter from a variety of

North American forests have respiration rates between

�2 and �8 nmol O2 s�1 g�1 (Burton et al., 2002), and

three Typha species have respiration rates ranging from

�1 to �7 nmol O2 s�1 g�1 (Matsui Inoue & Tsuchiya,

2008). Thus, A. dumosa and L. tridentata have greater

respiration rates than those reported for other woody

and herbaceous species. One possible explanation for

our observations of higher rates may be because the

time between root collection and respiration measure-

ment in our study was minimal (o20 min after harvest)

whereas time between collection and measurement for

other studies were up to 4 h (Pregitzer et al., 1998;

Burton et al., 2002).

Sample date

In the field experiment, both shrubs had reduced res-

piration rates at the end of the growing season in 2005

(Table 3, Fig. 2a and b). During 2005, soil water content

also was much lower than at the beginning of the year

(Fig. 2c). In this year, we found a significant, positive

relationship between soil moisture and root respiration

(result not shown). Although 2003 had a similar trend

as 2005, 2003 had too few data points to reliably conduct

a regression analysis. We also were not able to combine

data across years because differences among years in

other factors that affect root respiration (such as pre-

vailing soil temperatures, plant phenological stage at

the time of measurement, root age characteristics, etc.)

differed among years and because soil water and root

respiration were not always measured on the same

dates. Nonetheless, the relationship between soil water

and root activity is well documented in many systems

(e.g. Burton et al., 1998). It is important to note that

although we observed a decline in respiration rates of

L. tridentata and A. dumosa with low soil moisture, water

availability at this site is highly confounded with tem-

perature and phenological differences. To accurately

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determine the importance of soil moisture on root activ-

ity, a manipulative experiment should be implemented

with varying water availability throughout the season to

account for temperature and phenological effects.

Root diameter

Varying root diameters of A. dumosa and L. tridentata had

significantly different respiration rates (Table 3). Very fine

roots of both species had greater respiration rates than

fine roots, and this effect was not surprising given that

the finest roots are typically responsible for nutrient

uptake (Pregitzer et al., 1998; Desrochers et al., 2002).

However, when roots are classified by branching order,

clearer relationships between root form and function are

reported (e.g. Pregitzer et al., 2002). Indeed, when we

compared primary vs. lateral roots in our 4-week-old

L. tridentata seedlings, we found a trend toward greater

lateral root respiration (Table 1). Owing to our field

sampling technique, we could not always determine

branching order, although the majority of harvested very

fine roots appeared to be first branching order.

Root age

Root respiration rates were not significantly different

between age classes for L. tridentata but were for

A. dumosa, with older roots having lower respiration

rates than young roots (Table 3). Root nutrient uptake is

typically thought to decline with age (Eissenstat &

Yanai, 1997), which would decrease root respiration,

and grape, apple and citrus roots have all exhibited a

decline in root respiration with age (Bouma et al., 2001;

Volder et al., 2005). Although our results for A. dumosa

are consistent with these previous observations, it is not

clear why L. tridentata did not show similar trends.

Because of limited root availability and collection

feasibility, we defined root age as a categorical variable

(young or old) rather than numerical (exact age), with

the cutoff value of 1 month due to sampling constraints

(1 month was the approximate time interval between

root samplings). However, because 75% of L. tridentata

roots observed in the field with minirhizotrons survived

for over 1 year (S. Ferguson et al., unpublished data),

defining root age as older or younger than 1 month may

not have provided the resolution needed to detect root

age effects.

Conclusions

Our results from both the greenhouse and field experi-

ments suggest that root systems of L. tridentata and

A. dumosa have not altered carbon use for respiration

processes nor have they grown in size to compensate for

any nutrient limitations imposed by carbon fertilization.

Indeed, cumulative resin nitrogen and soil enzyme

activities are greater with elevated CO2 (Billings et al.,

2004; Jin & Evans, 2007), which indicate a change in

microbial community composition or activity with ele-

vated CO2 in the Mojave Desert. This carbon-limited

microbial community (Schaeffer et al., 2003) may be

stimulated by an increase in available carbon from litter

or root exudation. Increased aboveground productivity

with elevated CO2 (Smith et al., 2000; Nowak et al., 2004;

Housman et al., 2006) likely increases plant litter inputs

into the ecosystem (Weatherly et al., 2003). Root exu-

dation under elevated CO2 may also be altered, and

changes in the composition of root exudates could

consequently influence the heterotrophic communities

residing there (Jones et al., 2004). However, root exuda-

tion responses to elevated CO2 have not been investi-

gated for these Mojave Desert species and should be

investigated in future studies to clarify the potential for

plant-mediated shifts in soil carbon fluxes.

Acknowledgements

Funding for this research was provided by US DOE Office ofScience (DE-FG02-03ER63650 and DE-FG02-03ER63651) with addi-tional support from DOE National Nuclear Security Administra-tion/Nevada Operations Office, Brookhaven National Laboratory,and the Nevada Agricultural Experiment Station. We would like tothank Christina Wells, Vicki Longozo, Renee Richards, AndrewYoung, Dave Bryla, Iker Aranjuelo Michelana, Scot Ferguson, JamesKim, Eric Hoskins, and Christopher Holmes for help with the fieldand greenhouse studies and Kim Allcock and George Fernandezwith statistical analyses.

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