Tree Physiology 31, 391–401
doi:10.1093/treephys/tpr014
Research paper
Responses of sugar maple and hemlock seedlings to elevated
carbon dioxide under altered above- and belowground
nitrogen sources
Allyson S.D. Eller1,3,4, Krista L. McGuire2 and Jed P. Sparks1
1Ecology
and Evolutionary Biology Department, Cornell University, Corson Hall Rm E150, Ithaca, NY 14853, USA; 2Department of Biological Sciences, Barnard College, Columbia
University, 3009 Broadway, New York, NY 10027, USA; 3Current address: Cooperative Institute for Research in Environmental Sciences, University of Colorado, UCB 216,
Boulder, CO 80309-0216, USA; 4Corresponding author ([email protected])
Received September 16, 2010; accepted February 10, 2011; published online April 6, 2011; handling Editor Peter Millard
Various human-induced changes to the atmosphere have caused carbon dioxide (CO2), nitrogen dioxide (NO2) and nitrate
deposition (NO3−) to increase in many regions of the world. The goal of this study was to examine the simultaneous influence
of these three factors on tree seedlings. We used open-top chambers to fumigate sugar maple (Acer saccharum) and eastern
hemlock ( Tsuga canadensis) with ambient or elevated CO2 and NO2 (elevated concentrations were 760 ppm and 40 ppb,
respectively). In addition, we applied an artificial wet deposition of 30 kg ha−1 year−1 NO3− to half of the open-top chambers.
After two growing seasons, hemlocks showed a stimulation of growth under elevated CO2, but the addition of elevated NO2
or NO3− eliminated this effect. In contrast, sugar maple seedlings showed no growth enhancement under elevated CO2 alone
and decreased growth in the presence of NO2 or NO3−, and the combined treatments of elevated CO2 with increased NO2 or
NO3− were similar to control plants. Elevated CO2 induced changes in the leaf characteristics of both species, including
decreased specific leaf area, decreased %N and increased C:N. The effects of elevated CO2, NO2 and NO3− on growth were
not additive and treatments that singly had no effect often modified the effects of other treatments. The growth of both
maple and hemlock seedlings under the full combination of treatments (CO2 + NO2 + NO3−) was similar to that of seedlings
grown under control conditions, suggesting that models predicting increased seedling growth under future atmospheric
conditions may be overestimating the growth and carbon storage potential of young trees.
Keywords: Acer saccharum, carbon dioxide, global change, nitrate deposition, nitrogen dioxide, Tsuga canadensis.
Introduction
To predict the effects of future atmospheric change on plants,
most studies examine only single factors, but in order to generate realistic predictions for the future, it is important to conduct
multifactor experiments. When plants are exposed to simultaneous treatments, the responses are not always what would
have been predicted from single-treatment studies. In the present study, we used a factorial design to examine the single and
combined effects of elevated carbon dioxide (CO2), gaseous
nitrogen dioxide (NO2) and soil deposition of nitrate (NO3−).
We used this approach to explore instances where the combination of treatments may not be simply additive and to better
predict how plants are likely to respond to future changes in
atmospheric composition.
There is general scientific consensus that human activities,
particularly the burning of fossil fuels, are changing the chemistry of the Earth’s atmosphere and increasing global emissions of
CO2 and reactive nitrogen (N). Carbon dioxide emissions have
increased by 80% since 1970, and the global CO2 concentration
is increasing by 1.9 ppm per year (IPCC 2007). Between 1860
and 2000, the total amount of reactive N produced by human
processes increased by >1000% (from 15 to 165 Tg N year−1),
with reactive N from fossil fuel burning increasing by 2500%
(from 1 to 25 Tg N year−1; Galloway et al. 2003).
© The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
392 Eller et al.
Reactive N in the atmosphere can be deposited in the biosphere as wet deposition when N compounds are dissolved in
water and enter the ecosystem through precipitation or
through dry deposition when reactive N deposits directly on
surfaces. In the focus region of this study, northern lower
Michigan, ~60% of the total wet deposition of N is in the form
of NO3− (Pregitzer et al. 2008). In 2004 and 2005, NO3−
­deposition throughout the Midwestern USA ranged from 11 to
18 kg ha−1 year−1 (National Atmospheric Deposition Program,
NADP, http://nadp.sws.uiuc.edu), but some of the highest N
deposition sites in Europe receive as much as 60 kg N ha−1 year−1
(MacDonald et al. 2002).
Nitrogen dioxide is one component of dry deposition that is
increasing locally along roadways and around point sources. In
the Northeastern USA, ~50% of NOx (NO2 + NO) emissions
comes from vehicle emissions and another 25% comes from
the production of electricity. In urban areas in the USA, the
concentration of NO2 is typically 10–45 ppb (NASA Visible
Earth, http://visibleearth.nasa.gov), and 22–45% of Europeans
living in urban environments now experience background NO2
levels of >20 ppb (© EEA, Copenhagen 2008). At the study
site in northern Michigan, the background concentration of NO2
was typically <1 ppb (NO2 measured daily in control chambers;
data not shown).
Many studies have examined the influence of elevated CO2
on plant gas exchange and the growth of plants. Several review
papers and meta-analyses report that the majority of elevated
CO2 studies find at least a short-term increase in light-saturated
photosynthesis (Curtis and Wang 1998, Norby et al. 1999,
Long et al. 2004, Ainsworth and Long 2005, Ainsworth and
Rogers 2007) and a decrease in stomatal conductance in
response to elevated CO2 (Norby et al. 1999, Medlyn et al.
2001, Long et al. 2004, Ainsworth and Long 2005, Ainsworth
and Rogers 2007), although the magnitude of the variation in
response is often dependent on the functional group of the
plants examined and the growing conditions in the study
(Ainsworth and Long 2005, Ainsworth and Rogers 2007). The
observed increase in photosynthetic rates typically leads to an
increase in aboveground biomass (Curtis and Wang 1998,
Norby et al. 1999, Long et al. 2004, Ainsworth and Long 2005,
de Graaff et al. 2006), with the exception of plants whose
growth is limited by nutrients (Curtis and Wang 1998, Oren
et al. 2001) or water (Housman et al. 2006).
In addition to altering plant gas exchange and growth, CO2
has been shown to change the elemental ratios of tissues and
the allocation of biomass within the plant. In particular, most
studies find that elevated CO2 decreases the %N of leaf material (Curtis and Wang 1998, Norby et al. 1999, Yin 2002, Long
et al. 2004, Ainsworth and Long 2005, Körner et al. 2005,
Taub and Wang 2008), which can lead to an increase in the
ratio of carbon to nitrogen in plant biomass (C:N). Because
elevated CO2 provides more carbon substrate for ­photosynthesis
Tree Physiology Volume 31, 2011
and increases the C:N of leaves, many researchers have
hypothesized that plants should increase allocation to roots
because the extra carbon will increase limitation by other nutrients. However, few studies have actually seen an increase in
root:shoot ratio under elevated CO2 (Curtis and Wang 1998,
Norby et al. 1999, Zak et al. 2000).
Wet deposition of NO3− can affect photosynthesis and
growth in two opposing ways. Since N is limiting in many forested ecosystems (Vitousek and Howarth 1991), wet deposition of NO3− can alleviate N limitation, causing an increase in
foliar N content (e.g., Fenn et al. 1998, Magill et al. 2000, Yin
2002, Boggs et al. 2005, Xia and Wan 2008) and overall tree
growth (Pregitzer et al. 2008, Xia and Wan 2008). However,
long-term high deposition of N can lead to N saturation (Aber
et al. 1989). This occurs when there is an excess of N in the
ecosystem such that it affects the balance of soil processes
and leads to depletion of base cations (particularly calcium and
magnesium) and acidification of the soil (Fenn et al. 1998).
Typically it takes years of chronic N deposition to reach N saturation, although the length of time required to deplete soil cations and acidify the soil is strongly influenced by soil structure.
Nitrogen saturation of an ecosystem can lead to a decline in
plant growth and can increase tree mortality (e.g., Aber et al.
1989, McNulty et al. 1996).
Increased gaseous NO2 in the atmosphere can be directly
incorporated through foliage and can theoretically increase or
decrease photosynthesis and growth (Sparks 2009). When
NO2 enters plant leaves, it reacts with water and apoplastic
antioxidants such as ascorbate, producing nitrate and nitrite
(Zeevaart 1976, Murray and Wellburn 1985, reviewed by
Rennenberg and Gessler 1999). Nitrogen dioxide has also
been shown to contribute N to the formation of plant tissue,
suggesting that plants can use it as a source of N (Vallano and
Sparks 2007). Schmutz et al. (1995) and Siegwolf et al. (2001)
found significant increases in biomass under ~100 ppb NO2,
and as much as 15% of a plant’s N has been observed to come
directly from NO2 (Siegwolf et al. 2001, Vallano and Sparks
2007). Like increasing NO3− deposition, it is expected that in
an N-limited system, elevated NO2 could stimulate plant growth.
However, NO2 is also an oxidant, and when it enters plant
leaves it has the potential to react with cell membranes and
damage internal cellular structures. At very high concentrations
of NO2, investigators have found reduced plant growth [Rowland
et al. 1987 (300 ppb), Srivastava and Ormrod 1984 (200–
500 ppb in soil with >1 mM NO3− addition)] and increased
mortality [Srivastava 1992 (review), Qiao and Murray 1997
(300 ppb NO2)], although the responses tend to be species
specific.
One of the ecosystem level problems predicted to emerge
under elevated CO2 is progressive N limitation (Luo et al.
2004). Many have hypothesized that limitation by N will eventually constrain the growth enhancement caused by elevated
Response of sugar maple and hemlock to CO2 and N 393
CO2, and experimental evidence has shown that in many cases
when elevated CO2 is combined with increased soil N, the
increase in total biomass can be greater than when CO2 alone
is elevated (Curtis et al. 2000, Zak et al. 2000, Oren et al.
2001, de Graaff et al. 2006, Xia and Wan 2008). Plants that
accumulate more N are likely to increase the %N of leaf tissue
(Xia and Wan 2008), which may counteract the effect of CO2.
Increased N in the soil can decrease the allocation of biomass
to roots if the plant can get the same nutrients from a smaller
soil volume (Zak et al. 2000). If NO2 does not cause oxidative
damage, then it may provide an additional source of N and we
predict that plant responses to elevated NO2 may be similar to
those to increased NO3−.
In this study, we examined the single and combined effects
of elevated CO2, NO2 and increased wet deposition of NO3− on
two climax community species that are common in the
Northeastern USA, sugar maple, Acer saccharum, and eastern
hemlock, Tsuga canadensis, and predicted the following combinatorial responses.
(1) Additional N as either NO2 or NO3− will increase growth
under elevated CO2 by alleviating N limitation.
(2) Elevated CO2 will decrease the effects (both positive and
negative) of NO2 as a result of decreased stomatal conductance limiting NO2 entry into leaves.
(3) The effects of NO2 will be less pronounced under increased
NO3− because the magnitude of additional N from NO2 will
be small compared with that from NO3−.
Methods
Site description
This study was conducted in an open field at the University of
Michigan Biological Station near Pellston, MI, USA (45°33′14″N,
84°47′4″W). Over the 2-year study period, the average high
and low June–August temperatures at the site were 23 and
13 °C, respectively. The average monthly temperatures in
2004 were typical of the previous 10 years, and while 2005
had a warmer than usual June, the rest of the summer was typical. In order to reduce the ambient light level and approximate
the understory environment, a shade cloth was erected 2 m
above the entire site, reducing the incoming light by 50%. The
shade cloth was porous and allowed at least some natural precipitation to pass through. The total summer precipitation in
2004 and 2005 was 20.7 and 27 cm, respectively, and the
plants in the chambers were watered every 3 days (if there
was no natural precipitation) to avoid drought stress.
Plant material and chambers
Bare-rooted seedlings of hemlock (T. canadensis) and sugar
maple (A. saccharum) were purchased from Pikes Peak Nursery
(Penn Run, PA, USA). Pikes Peak Nursery propagates sugar
maple and hemlock from seed, and they are planted in an open
field where they receive ambient CO2 and are given no soil
amendments. The seedlings were 3–5 years old and 45–60 cm
tall when planted into the experiment. In May 2004, two seedlings of each species were randomly assigned to each rootbox
and two rootboxes were placed under each chamber. In
September 2004, the seedlings from one rootbox of each
chamber were harvested. The harvested rootboxes were
placed back in the ground, and in May 2005 new seedlings
were planted. In all cases except the gas exchange data (where
the same individuals were measured in 2004 and 2005),
plants designated as ‘year 1’ were planted and harvested in
2005 while those designated as ‘year 2’ were planted in 2004
and harvested in 2005.
Rootboxes were prepared by drilling ~30–2.5 cm holes into
the bottoms of plastic storage tubs (82 × 52 × 42 cm). The
holes allowed for free drainage of water through the boxes.
The boxes were buried so that the top of each box was level
with the surrounding soil surface. The boxes were filled with
sand and covered with 10 cm of topsoil (from local sources) to
establish mycorrhizal associations and a microbial community
similar to that of the local forests. This sand/soil layering mimicked the soil structure in the surrounding forests.
One open-top chamber (0.8 m × 0.8 m × 1 m) was placed
over each pair of rootboxes. The chamber frames were made of
1/2 inch PVC pipe and were wrapped with transparent 0.8 mm
PVC film. Fans encased in metal blower boxes were connected
to a perforated ring of PVC that was placed at the bottom of the
chamber. The bulk flow from the blower box through the chamber was 600–700 l min-1, resulting in a turnover time of <2 min.
A smoke test showed that the chambers became fully mixed in
<10 s. The temperature inside the chambers was typically 3 °C
warmer than the surrounding environment, but was uniform
between all the chambers. The chambers and shade cloth were
removed from October through April so that there were no differences in winter temperature or snow depth.
A total of 80 chambers were used. All the chambers were
assigned to a block (10 total blocks) based on their location in
the field in order to minimize the effects of possible light, wind
and moisture differences across the field. Within each block,
each chamber was randomly assigned to one of eight possible
treatment combinations: control (ambient CO2, ambient NO2,
no soil NO3− addition), elevated CO2, elevated NO2, high NO3−,
elevated CO2 + elevated NO2, elevated CO2 + high NO3−, elevated NO2 + high NO3− and elevated CO2 + ­elevated NO2 + high
NO3−.
Treatments
Carbon dioxide was purchased from Airgas (Charlevoix, MI,
USA) as a liquid and NO2 was purchased in 10,000 ppm tanks
from Scott-Marin Specialty Gas (Riverside, CA, USA). The gas
from each tank was delivered to a manifold block where it was
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394 Eller et al.
split into 40 lines, and the flow of each line was controlled by a
needle valve and flowmeter (Aalborg, Orangeburg, NY, USA).
Opaque black Teflon (PTFE) tubing was used for all the NO2
lines and Poly Flo tubing (J.F. Good Company, Sebring, OH,
USA) was used for the CO2 lines. Return lines placed in each
chamber were used to bring air from the chamber to a solenoid
system that automatically sampled each chamber every 4 h. An
infrared gas analyzer (LI 6252; Li-Cor, Lincoln, NE, USA) was
used for analysis of CO2 concentration and a chemiluminescence analyzer (CLD 760; EcoPhysics Duernten, Switzerland)
fitted with a NO2 converter (PLC 660; Ecophysics) was used for
measurement of NO2 and NO concentrations. Both analyzers
were calibrated weekly using sequential dilution of certified calibration tanks (Scott-Marrin Specialty Gas, Riverside, CA, USA).
The elevated CO2 treatment began on 20 June 2004 and 13
June 2005 and ended on 15 September 2004 and 22 August
2005 when the plants were harvested. The elevated NO2 treatment was delayed in 2004 and did not start until 10 July, but
otherwise had the same beginning and ending dates as elevated CO2. In both years, the fumigation treatments began after
leaf-out. In 2004, the treatments ended 2 weeks before the
leaves began to senesce. Elevated CO2 chambers were set to
760 ppm CO2, elevated NO2 chambers received 40 ppb NO2,
and the treatments were applied between 7 a.m. and 7 p.m.
daily. The concentration of each gas in each chamber was
checked daily and adjusted to the target if required. NO2 concentrations were within 10 ppb of the target >90% of the time
and CO2 concentrations were within 50 ppm of the target
>90% of the time. The NO concentration in elevated NO2
chambers was typically 3–7 ppb. The ambient CO2 concentration was 365 ppm and ambient NO2 and NO concentrations
were both <1 ppb. The ozone concentration in each chamber
was measured three times each summer, and we found that
the addition of NO2 did not increase ozone levels above
­ambient (data not shown).
Half the chambers were given additional soil N in the form of
NaNO3 at a rate of 30 kg N ha−1 year−1. Each year, solid fertilizer was applied and immediately watered in four times each
summer at 2 week intervals beginning in 22 June 2004 and 14
June 2005. In both years, the initial NO3− addition was after
bud-break, and the repeated applications were designed to
maintain high NO3− in the soil throughout the growing season.
Photosynthesis measurements
The Li-Cor LI-6400 portable gas exchange system (Li-Cor)
was used for all gas exchange measurements. Photosynthesis
and stomatal conductance were measured in July and August in
both years. In both years, we only measured the maple seedlings that were planted in 2004, meaning that the gas exchange
data for the first-year treatment were collected in 2004 on
seedlings planted in 2004. The second season data were collected on the same seedlings in 2005. For all the other response
Tree Physiology Volume 31, 2011
variables, the data presented for first-year seedlings are from
seedlings that were planted in 2005. Due to time constraints
and equipment availability, we were unable to measure gas
exchange on the hemlock seedlings.
Photosynthesis was measured at two CO2 concentrations for
each seedling. First, each seedling was measured at the CO2
concentration under which it was growing (i.e., seedlings
grown under ambient CO2 were measured at 380 ppm CO2
and those grown under elevated CO2 were measured at
760 ppm CO2) and then each seedling was measured at
760 ppm. We used the first measurements to look for absolute
differences in gas exchange. The second measurements were
used to look for acclimation of photosynthesis to elevated CO2
by comparing the data collected on plants grown under elevated CO2 with those on plants grown at ambient CO2 and
instantaneously exposed to elevated CO2.
Growth and allocation measurements
At the time of planting, 10 seedlings of each species were harvested and the height, stem diameter and total dry biomass
were measured. A multiple linear regression model was developed using height and stem diameter to predict initial dry biomass and create an estimate of the initial dry biomass for each
seedling planted in the study. Estimated initial biomass was
used as a covariate in all subsequent analyses of biomass.
In September 2005, all seedlings were harvested by removing the rootboxes from the ground and placing the soil and
seedlings onto a 2 mm mesh screen to rinse soil from the
roots. Once clean, the roots, stems and leaves were separated,
the roots frozen, and the leaves pressed. The separated seedlings were transported to Cornell University in Ithaca, NY, where
all the plant tissues were dried for 3 days at 50 °C and weighed
to determine dry mass (the same procedure was used during
the plant harvest in 2004). The mass values reported for total
root production include both woody and fine root fractions.
The total leaf area of maple seedlings was determined using
a leaf area meter (LI-3100, Li-Cor). Leaf area was divided by
total leaf biomass to determine specific leaf area (SLA). To
determine the SLA of hemlock seedlings, only needles that
were produced in 2005 were used. The area of hemlock needles was determined by photographing several needles on a
background of known area. Using Adobe Photoshop software
(Adobe Systems, Inc., San Jose, CA, USA), we calculated the
percentage of background that was covered with needles. The
photographed leaves were then weighed to determine SLA.
Dried leaf tissue was ground and %C and %N were
­determined using an Elemental Analyzer (FlashFA 1112;
ThermoFinnegan, Pittsburg, PA, USA).
Statistical analysis
In this randomized block design there were 10–20 replicates
of each species in each treatment, depending on seedling
Response of sugar maple and hemlock to CO2 and N 395
­ ortality. Most seedling mortality occurred shortly after transm
plantation to the field and was not related to treatment (data
not shown). Treatment means were compared using mixedmodel analysis of variance and analysis of covariance techniques with pairwise comparisons. All the analyses of mass
included estimated initial biomass as a covariate and were
transformed by taking the square root in order to eliminate
increasing variance in the residuals; total leaf area was also
analyzed after a square root transformation. An α of 0.05 was
used in all tests. All statistical analyses were completed using
SAS statistical software (SAS Version 9.1.3; SAS Institute, Inc.,
Cary, NC, USA) and figures were generated in SigmaPlot (SPSS
Science, Chicago, IL, USA).
Results
Biomass
Elevated CO2 increased the biomass in sugar maple seedlings
grown for 1 year under treatment compared with the control
(Figure 1a). However, this change diminished after 2 years
of treatment (Figure 1b). In hemlock, plants grown under
­elevated CO2 showed increased total plant biomass compared
with the control after 2 years of treatment (Figure 1c).
Biomass was not assessed in hemlock after 1 year of treatment. In both species the CO2 effect was eliminated by the
presence of elevated NO2, and in hemlock the addition of soil
NO3− also eliminated the CO2 effect. In contrast, after 2 years
Figure 1. Mean total, leaf, stem and root dry mass (g) with error bars indicating ±SE. Diagonal lines, open bars and cross-hatch bars indicate maple
seedlings after 1 year of treatment, maple seedlings after 2 years of treatment and hemlock seedlings after 2 years of treatment, respectively.
aNO3− = ambient NO3− deposition, eNO3− = 30 kg ha−1 year−1 NO3− deposition (high NO3−), aNO2 = ambient NO2 and eNO2 = 40 ppb NO2. Asterisks
indicate a significant difference between the ambient and elevated CO2 pairs (α = 0.05). Open circles indicate a significant difference between that
treatment and the control.
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396 Eller et al.
of treatment the total biomass of sugar maple seedlings
showed no response to elevated CO2 when NO2 and NO3−
were ambient (Figure 1b). Further, the addition of NO2 or
NO3− singly reduced total biomass, and the combination of
elevated CO2 and either NO2 or NO3− resulted in biomass not
different from control plants.
Allocation
The biomass of hemlock seedlings under elevated CO2
increased in leaf, stem and root tissue. This increase was uniform across tissue types (Figure 1f, i and l) and did not usually
alter the allocation of biomass (Table 1). The only treatment
that showed altered biomass allocation in hemlock was the
combination of elevated CO2 and soil NO3−, which caused a
decrease in the allocation of biomass to leaf tissue (Table 1).
In sugar maple, the duration of treatment altered the treatment effects on root:shoot ratio. Elevated CO2 caused a
decrease in root:shoot ratio after the first year of treatment,
and after 2 years the addition of N as either NO2 or NO3−
caused a decrease in root:shoot ratio (Table 1). A decrease in
root:shoot ratio caused by a single year of elevated CO2 was
observed in every ambient CO2/elevated CO2 comparison
except in NO2 + NO3− vs. CO2 + NO2 + NO3− (P = 0.08). After 2
years, plants exposed to elevated CO2 showed increased leaf
biomass compared with control, but only under ambient NO2,
and the increase did not significantly change the root:shoot
ratio (Table 1). Although neither NO2 nor NO3− had an effect on
root:shoot ratio after only 1 year, after 2 years the addition of
N as either NO2 or NO3− led to a reduction in root biomass
(Figure 1k) that decreased the root:shoot ratio (Table 1) and
tended to reduce total biomass (Figure 1b).
Gas exchange
Gas exchange characteristics were only measured in sugar
maple, and after one season under treatment there was no
evidence of photosynthetic acclimation to elevated CO2 (Table 2).
When seedlings were measured under elevated CO2, there
were no differences in photosynthetic rates between those
grown in elevated CO2 and those grown in ambient CO2. When
photosynthetic rates were measured at the treatment level CO2
concentration, they were generally higher in the seedlings in
the elevated CO2 group than in their ambient CO2 counterparts;
however, the difference was only significant in the absence of
NO2 or NO3− (NO2 vs. NO2 + CO2, P = 0.08; NO3− vs.
NO3− + CO2, P = 0.17; and NO2 + NO3− vs. CO2 + NO2 + NO3−,
P = 0.08).
Photosynthetic rates were generally lower in the second year
compared with the first, and there were no differences in photosynthetic rate between treatments when the seedlings were
measured at the CO2 concentration under which they were
growing (Table 2). In addition, when seedlings were measured
at 760 ppm CO2, those grown under elevated CO2 had lower
photosynthetic rates than their ambient CO2 counterparts
(P < 0.05) in all the treatments except in the NO3− addition and
CO2 + NO3− treatments (P = 0.06).
Elevated CO2 caused decreased stomatal conductance, but
it took 2 years for the effect to be seen across all treatments
(Table 2). After the first year, only CO2 + NO2 and CO2 + NO3−
Table 1. Seedling root:shoot ratio and the % of total biomass located in each tissue type in sugar maple after 1 and 2 years of treatment and hemlock exposed to 2 years of treatment. Standard error for each mean is shown in parentheses. Superscript letters represent pairwise comparisons
where the difference between means that do not share a letter is statistically significant (α = 0.05). Treatments are shown in column headings
where eCO2 = 760 ppm CO2, eNO2 = 40 ppb NO2 and eNO3− = soil NO3− added at 30 kg ha−1 year−1.
Control
Maple
year 1
Maple
Year 2
eCO2
eNO2
Root:shoot 0.87bc (0.07) 0.66a (0.04)
ratio
eNO2 + eCO2
21.08a (1.49) 29.86c (1.83) 21.11a (1.39)
%Stem
33.85bc
%Root
45.06bc
(1.48)
(2.14)
39.22a
Root:shoot 1.32a (0.05)
ratio
eNO3− + eCO2 eNO2 + eNO3− eNO2 + eNO3− + eCO2
0.89bc (0.08) 0.82bc (0.04) 0.82bc (0.05) 0.65a (0.04) 0.93c (0.06)
%Leaf
30.79ab
eNO3−
0.78ab (0.07)
23.56ab (1.56) 19.93a (1.65) 27.72bc (1.51) 20.61a (1.17) 27.58bc (1.89)
(1.63)
33.41abc
(2.23) 31.92ab (1.16) 35.95c (1.89) 34.04bc (1.43) 32.27ab (1.58) 29.94a (1.56)
(1.45)
45.55bc
(1.96) 44.55bc (1.16) 44.18bc (1.67) 38.88a (1.62) 47.12c (1.70)
1.20ab (0.07) 1.06b (0.07)
1.06b (0.06)
1.15b (0.07)
1.10b (0.05)
1.11b (0.06)
42.51ab (2.09)
1.16b (0.06)
%Leaf
21.45a (0.57) 23.55b (0.77) 23.67b (0.77) 24.04b (1.00) 23.74b (0.77) 27.01c (0.93) 24.14b (0.86) 25.25bc (1.05)
%Stem
21.91abc (1.01) 22.49abc (0.95) 25.75d (1.36) 25.04d (1.27) 23.61bcd (0.97) 21.00a (0.87) 23.96cd (0.88) 21.52ab (0.94)
%Root
56.57a (1.00) 53.88ab (1.43) 50.54b (1.88) 50.78b (1.68) 52.63b (1.47) 51.96b (1.16) 51.90b (1.37) 53.25b (1.35)
Hemlock Root:shoot 0.40a (0.04)
Year 2
ratio
0.43a (0.05)
0.42a (0.03)
0.39a (0.02)
0.43a (0.02)
0.44a (0.03) 0.43a (0.03)
0.44a (0.02)
%Leaf
29.52a (1.78) 29.34ab (2.78) 26.84ab (0.91) 26.67abc (1.40) 26.28bc (0.97) 23.75c (1.91) 28.02ab (1.05) 28.80ab (0.73)
%Stem
42.21a (2.35) 41.05ab (3.20) 44.05ab (1.52) 45.41ab (1.79) 44.17ab (1.52) 45.96b (2.89) 42.06b (1.67) 40.89ab (1.21)
%Root
28.21a (1.73) 29.59ab (2.19) 29.13ab (1.30) 27.96a (1.01) 29.54ab (1.10) 31.66b (1.56) 29.88ab (1.43) 30.30ab (1.02)
Tree Physiology Volume 31, 2011
Response of sugar maple and hemlock to CO2 and N 397
Table 2. Mean photosynthetic rate measured at treatment level CO2 (Atreatment) (µmol CO2 m−2 s−1), photosynthetic rate measured at 760 ppm CO2
(A760) (µmol CO2 m−2 s−1) and stomatal conductance measured at treatment level CO2 (gs treatment) (mmol H2O m−2 s−1)for sugar maple seedlings
exposed to 1 and 2 years of treatment. Standard error for each mean is shown in parentheses. Superscript letters represent pairwise comparisons
where the difference between means that do not share a letter is statistically significant (α = 0.05). Treatments are shown in column headings
where eCO2 = 760 ppm, eNO2 = 40 ppb NO2 and eNO3− = soil NO3− added at 30 kg ha−1 year−1.
Maple
year 1
Atreatment
A760
gs treatment
Maple
year 2
Atreatment
A760
gs treatment
Control
eCO2
eNO2
eNO2 + eCO2
eNO3−
eNO3− + eCO2
eNO2 + eNO3−
eNO2 + eNO3− + eCO2
6.45bc
(0.99)
8.45ab
(1.67)
0.093a
(0.022)
4.45ab
(0.57)
6.59a
(0.61)
0.047ab
(0.009)
9.92a
(0.88)
9.92a
(0.88)
0.066ab
(0.007)
4.95ab
(1.10)
5.07bc
(1.10)
0.023c
(0.005)
6.37bc
(0.29)
7.90ab
(0.73)
0.086a
(0.011)
3.88ab
(0.65)
5.50ab
(0.25)
0.046ab
(0.007)
9.05ab
(2.27)
9.05ab
(2.27)
0.051bcd
(0.020)
3.63ab
(0.18)
3.79cd
(0.18)
0.016c
(0.002)
6.23bc
(0.36)
8.91ab
(0.27)
0.065abc
(0.010)
3.60b
(0.67)
4.88bcd
(0.49)
0.060ab
(0.026)
8.11ab
(0.67)
8.18ab
(0.67)
0.035d
(0.006)
3.51b
(0.54)
3.58d
(0543)
0.018c
(0.003)
5.18c
(0.59)
5.74b
(1.31)
0.054abcd
(0.011)
4.22ab
(0.75)
6.08ab
(0.99)
0.071a
(0.018)
7.66ac
(1.39)
7.66ab
(1.39)
0.044bcd
(0.009)
5.07a
(0.93)
5.24ab
(0.93)
0.029bc
(0.009)
had lower conductance than their ambient CO2 counterparts.
However, after 2 years of treatment all plants grown under elevated CO2 had lower conductance compared with their ambient
CO2 counterparts.
Leaf characteristics
Total seedling leaf area was influenced by elevated CO2 after 1
year of fumigation, but the effects were eliminated after the
second year. Elevated CO2 increased the total seedling leaf
area in every elevated CO2/ambient CO2 pair except in NO2 vs.
CO2 + NO2 (Figure 2a). After 2 years, no treatment had an effect
on total seedling leaf area (Figure 2b).
Specific leaf area decreased under elevated CO2 in most
cases regardless of species or treatment length (Figure 2c–e).
The two exceptions were maples exposed to 2 years of elevated CO2 with ambient NO2 and NO3− and hemlocks exposed
to the combined treatment of elevated CO2 + elevated
NO2 + elevated NO3−.
Elevated CO2 decreased leaf %N and increased C:N in sugar
maple under all treatment cases except one (Figure 3a, b, g
and h). Seedlings grown under elevated CO2 and soil NO3−
showed higher foliar %C than leaves from the elevated NO3−
treatment and C:N was not significantly different between the
ambient and elevated CO2 groups (P = 0.09). The elemental
Figure 2. Total leaf area (cm2) and SLA (cm2 g−1). Error bars indicate ±SE. Diagonal lines, open bars and cross-hatch bars indicate sugar maple
seedlings after 1 and 2 years of treatment and hemlock seedlings after 2 years of treatment, respectively. aNO3− = ambient NO3− deposition,
eNO3− = 30 kg ha−1 year−1 NO3− deposition (high NO3−), aNO2 = ambient NO2 and eNO2 = 40 ppb NO2. Asterisks indicate a significant difference
between ambient and elevated CO2 pairs and circles indicate a significant difference between that treatment and the control.
Tree Physiology Online at http://www.treephys.oxfordjournals.org
398 Eller et al.
composition of hemlock leaves was also altered by both elevated CO2 and higher soil NO3− (Figure 3c, f and i). Elevated
CO2 decreased foliar %N and %C, and increased C:N. In contrast, increased soil NO3− caused an increase in foliar N and a
corresponding decrease in the C:N of hemlock leaves. The
combination of increased CO2 and soil NO3− balanced the
changes in C and N such that the %N and C:N of seedlings
in these treatments were similar to those of seedlings in the
control treatment.
Discussion
The benefit of multifactor experiments is the increased ability
to predict plant performance in ecosystems experiencing multiple environmental changes, without assuming that the effects
of the individual changes will be additive. In this study, we
found that both sugar maple and hemlock seedlings growing in
environments with elevated CO2 + elevated NO2 + high NO3−
had biomass accumulation rates and allocation patterns that
were similar to the control. If the combination of individual
treatments had been purely additive, then sugar maples in the
elevated CO2 + elevated NO2 + high NO3− treatment would
have exhibited decreased growth relative to the control while
hemlocks would have exhibited enhanced growth. The inability
to predict non-additive treatment effects is a well-known
­limitation of many models, and results like those presented
here should inform future modeling efforts.
After 2 years, the photosynthetic activity of sugar maple
seedlings in this study had acclimated to elevated CO2.
Seedlings grown under elevated CO2 did not have higher rates
of photosynthesis measured at treatment level CO2 (i.e., plants
grown at ambient CO2 were measured at 380 ppm CO2 while
those grown at 760 ppm CO2 were measured at 760 ppm
CO2), but when photosynthesis was measured at 760 ppm CO2
for all the seedlings, those grown at ambient CO2 had significantly higher rates of photosynthesis than those grown at
760 ppm CO2. Photosynthetic acclimation to CO2 has been
previously reported in sugar maple [Kubiske et al. 2002,
Karnosky et al. 2003 (results from a FACE study)] and as a
common, although variable, response in FACE studies on a
variety of species (Ainsworth and Long 2005). Although we
cannot be sure whether the photosynthetic acclimation we
Figure 3. Elemental analysis of leaf tissue including %N, %C and leaf C:N with error bars indicating ±SE. Diagonal lines, open bars and cross-hatch
bars indicate sugar maple seedlings after 1 and 2 years of treatment and hemlock seedlings after 2 years of treatment, respectively. aNO3− = ­ambient
NO3− deposition and eNO3− = 30 kg ha−1 year−1 NO3− deposition (high NO3−), aNO2 = ambient NO2 and eNO2 = 40 ppb NO2. Different letters indicate a significant difference between means (α = 0.05).
Tree Physiology Volume 31, 2011
Response of sugar maple and hemlock to CO2 and N 399
observed is due to a reduction in Rubisco, we saw a clear
reduction in leaf %N, which tends to be reported along with
reduced Rubisco in elevated CO2 studies (Ainsworth and Long
2005) and is strongly correlated with photosynthetic rate in
both ambient and elevated CO2, especially within community
types (meta-analysis by Peterson et al. 1999). Photosynthetic
acclimation remains an important physiological mechanism to
consider when predicting future carbon fixation by plants.
In general, sugar maple growth appears to be less influenced
by elevated CO2 than growth in other species. A number of
studies that focused on sugar maple also report no increase in
growth under elevated CO2 at ambient temperatures and without NO3− fertilization (Reid and Strain 1994, Kinney and
Lindroth 1997, Gaucher et al. 2003, Karnosky et al. 2003,
Norby et al. 2007). The level of photosynthetic acclimation
observed in this study makes it unsurprising that we saw no
growth enhancement under elevated CO2 since the plants were
not fixing extra carbon despite the additional CO2 in the environment. Many studies predict that nutrient limitations in ecosystems will ultimately limit the growth response of forests to
elevated CO2 (Körner 2006, Reich et al. 2006a, 2006b,
Millard et al. 2007) and that N in particular will become progressively limiting through time (Luo et al. 2004, Hungate et al.
2006). Sugar maple growth in this study does not appear to
be carbon limited, but since the addition of NO3− did not stimulate growth (with or without elevated CO2) N limitation also
seems unlikely. We did not measure the concentrations of any
macro- or micronutrients in the soil, and sugar maple is known
to be particularly sensitive to nutrient imbalances (St. Clair
et al. 2008); hence it seems likely that the nutrient-poor sandy
soils in which our seedlings grew were deficient in some
unmeasured nutrient. Since sugar maple is an economically
and ecologically important species in the Northeastern USA, it
is important to consider whether the lack of growth enhancement in seedlings grown under elevated CO2 seen here and
elsewhere may decrease its competitive ability in the future.
Both NO2 and NO3− had the same effect on sugar maple
seedling growth: singly or together they reduced the total root
biomass of seedlings, leading to a reduction in total seedling
biomass and a decrease in root:shoot ratio. A relative reduction
in root growth is a commonly reported response to high soil
NO3− (e.g., Zhang and Forde 1998, Stitt 1999, Zhang and
Forde 2000, Zak et al. 2000, Xia and Wan 2008), and the
response may be caused by a build-up of NO3− in the shoot
acting as a signaling molecule that reduces root growth
(Scheible et al. 1997). Declines in root growth under elevated
NO2 have been previously reported, but at extremely high NO2
concentrations (5000 ppb; Srivastava et al. 1994). In one of a
very small number of studies using woody plants, a fumigation
period longer than 1 month, and high, but not extreme, concentrations of NO2 (80–135 ppbv), Siegwolf et al. (2001)
found that elevated NO2 increased total plant growth, but also
significantly decreased the root:shoot ratio. Since NO2 becomes
NO3− and NO2− after entering the apoplast of leaves, it may
have caused an increase in shoot NO3− that resulted in
decreased root ­production. Although neither NO3− nor NO2
caused an increase in leaf %N, the rise in leaf NO3− that was
associated with decreased root:shoot ratio in the Scheible
et al. (1997) study was 100–200 µmol NO3− gFW−1, which
would only increase leaf %N by 0.014–0.028%: a change
that is not detectable under most leaf elemental analysis
techniques.
The addition of elevated CO2 decreased the reduction in root
growth in sugar maples caused by NO2 and NO3−. The decrease
in stomatal conductance caused by elevated CO2 likely
decreased the entry of both NO2 and NO3− into the plant,
thereby reducing the amount of each compound entering the
plant. NO2 entry into leaves is primarily controlled by stomatal
conductance (Eller and Sparks 2006) and a reduction in stomatal conductance would result in a stepwise reduction in NO2
entering the plant. Further, reductions in stomatal conductance
are coupled with reductions in transpiration, which is the primary driver of the bulk movement of water from the soil through
the plant. Because NO3− enters plant roots via the bulk flow of
water from the soil, a reduction in transpiration would, at least
partially, reduce the amount of NO3− entering the plant via the
roots (Nye and Tinker 1977, Lambers et al. 1998). These two
mechanisms in concert likely decreased total nitrate entry into
the plant, suggesting that the primary influence of elevated CO2
in these treatments was to minimize the effects of N addition.
Despite being an important member of the northern hemlock–hardwood forest communities, very little research has
been done to determine how eastern hemlock will respond to
elevated CO2. Of the two studies found in the literature, one
pooled the responses of a number of species (Bauer et al.
2001), making it difficult to determine the specific responses
of hemlock. The other study conducted by Godbold et al.
(1997) found no significant changes in total or belowground
biomass of hemlock grown under elevated CO2. In contrast, we
did observe increased biomass in hemlock, but only when both
NO2 and NO3− were ambient. It is not clear why additional N
might eliminate CO2 growth enhancement, particularly when
neither NO2 nor NO3− had an effect on growth under ambient
CO2 conditions. If elevated CO2 decreased stomatal conductance, we would expect NO2 and NO3− to be less effective as an
N source under elevated CO2. The observed synergism between
elevated CO2 and additional N supply is unresolved, but is a
surprising result observed only because of the multifactorial
nature of this experiment, and should be investigated further.
Even in the absence of growth effects, elevated CO2 influenced foliar elemental composition and SLA in both hemlock
and sugar maple seedlings. In both species, elevated CO2
caused a decrease in leaf %N and an increase in C:N in every
treatment to which CO2 was added. These findings are in
Tree Physiology Online at http://www.treephys.oxfordjournals.org
400 Eller et al.
agreement with the majority of the studies that have examined
the effects of elevated CO2 (e.g., Curtis and Wang 1998, Norby
et al. 1999, King et al. 2001, Yin 2002, Ainsworth and Long
2005). In the treatments where NO3− deposition was high, the
combination of high CO2 and high NO3− balanced the C:N of
leaves such that the leaves of seedlings receiving both elevated
CO2 and high NO3− had C:N ratios similar to those of the
control.
The combinatorial nature of this experiment enabled us to
observe plant responses to conditions that may be more representative of future environmental conditions. We found that
simultaneous increases in CO2, NO2 and soil NO3− addition
caused plants to have growth rates and leaf C:N ratios similar to
those of plants in the control treatment, even though the application of single treatments had significant effects. This observation has significant ramifications for future predictions of plant
growth and performance based on single-factor experiments.
Acknowledgments
The authors would like to thank those who contributed to the
construction and execution of the fieldwork: Kathleen Bachynski,
Steve Bertman, Joseph Bump, Mary Anne Carroll, Peter Curtis,
Jessie Knapp, Carmody McCalley, Luke Spaete, Kimberlee
Sparks, Richard Spray, C. Anthony Sutterly, Nancy Tuchman
and the staff of the University of Michigan Biological Station.
Funding
Funding for this project was provided by the National Science
Foundation through the University of Michigan Biological
Station IGERT Program in Biosphere-Atmosphere Research
and Training, the Cornell IGERT program in Biogeochemisty
and Environmental Biocomplexity, the Doctoral Disseration
Improvement grant (DEB A61-8428) awarded to A.S.D.E. and
the Ecosystems Studies Grant (DEB-0237674) awarded to
J.P.S. Additional funding came from the Andrew W. Mellon
Foundation and the University of Michigan Biological Station.
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