Abstract

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.

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).

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 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 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 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 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 mortality. Most seedling mortality occurred shortly after transplantation to the field and was not related to treatment (data not shown). Treatment means were compared using mixed-model 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 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.

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 ha1 year1 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.

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 ha1 year1 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.

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).

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 eCO2 eNO2 eNO2 + eCO2 eNO3 eNO3 + eCO2 eNO2 + eNO3 eNO2 + eNO3 + eCO2 
Maple year 1 Root:shoot ratio 0.87bc (0.07) 0.66a (0.04) 0.89bc (0.08) 0.82bc (0.04) 0.82bc (0.05) 0.65a (0.04) 0.93c (0.06) 0.78ab (0.07) 
 %Leaf 21.08a (1.49) 29.86c (1.83) 21.11a (1.39) 23.56ab (1.56) 19.93a (1.65) 27.72bc (1.51) 20.61a (1.17) 27.58bc (1.89) 
 %Stem 33.85bc (1.48) 30.79ab (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) 
 %Root 45.06bc (2.14) 39.22a (1.45) 45.55bc (1.96) 44.55bc (1.16) 44.18bc (1.67) 38.88a (1.62) 47.12c (1.70) 42.51ab (2.09) 
Maple Year 2 Root:shoot ratio 1.32a (0.05) 1.20ab (0.07) 1.06b (0.07) 1.06b (0.06) 1.15b (0.07) 1.10b (0.05) 1.11b (0.06) 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 Year 2 Root:shoot ratio 0.40a (0.04) 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) 
  Control eCO2 eNO2 eNO2 + eCO2 eNO3 eNO3 + eCO2 eNO2 + eNO3 eNO2 + eNO3 + eCO2 
Maple year 1 Root:shoot ratio 0.87bc (0.07) 0.66a (0.04) 0.89bc (0.08) 0.82bc (0.04) 0.82bc (0.05) 0.65a (0.04) 0.93c (0.06) 0.78ab (0.07) 
 %Leaf 21.08a (1.49) 29.86c (1.83) 21.11a (1.39) 23.56ab (1.56) 19.93a (1.65) 27.72bc (1.51) 20.61a (1.17) 27.58bc (1.89) 
 %Stem 33.85bc (1.48) 30.79ab (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) 
 %Root 45.06bc (2.14) 39.22a (1.45) 45.55bc (1.96) 44.55bc (1.16) 44.18bc (1.67) 38.88a (1.62) 47.12c (1.70) 42.51ab (2.09) 
Maple Year 2 Root:shoot ratio 1.32a (0.05) 1.20ab (0.07) 1.06b (0.07) 1.06b (0.06) 1.15b (0.07) 1.10b (0.05) 1.11b (0.06) 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 Year 2 Root:shoot ratio 0.40a (0.04) 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) 

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).

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.

  Control eCO2 eNO2 eNO2 + eCO2 eNO3 eNO3 + eCO2 eNO2 + eNO3 eNO2 + eNO3 + eCO2 
Maple Atreatment 6.45bc 9.92a 6.37bc 9.05ab 6.23bc 8.11ab 5.18c 7.66ac 
year 1  (0.99) (0.88) (0.29) (2.27) (0.36) (0.67) (0.59) (1.39) 
 A760 8.45ab 9.92a 7.90ab 9.05ab 8.91ab 8.18ab 5.74b 7.66ab 
  (1.67) (0.88) (0.73) (2.27) (0.27) (0.67) (1.31) (1.39) 
 gs treatment 0.093a 0.066ab 0.086a 0.051bcd 0.065abc 0.035d 0.054abcd 0.044bcd 
  (0.022) (0.007) (0.011) (0.020) (0.010) (0.006) (0.011) (0.009) 
Maple Atreatment 4.45ab 4.95ab 3.88ab 3.63ab 3.60b 3.51b 4.22ab 5.07a 
year 2  (0.57) (1.10) (0.65) (0.18) (0.67) (0.54) (0.75) (0.93) 
 A760 6.59a 5.07bc 5.50ab 3.79cd 4.88bcd 3.58d 6.08ab 5.24ab 
  (0.61) (1.10) (0.25) (0.18) (0.49) (0543) (0.99) (0.93) 
 gs treatment 0.047ab 0.023c 0.046ab 0.016c 0.060ab 0.018c 0.071a 0.029bc 
  (0.009) (0.005) (0.007) (0.002) (0.026) (0.003) (0.018) (0.009) 
  Control eCO2 eNO2 eNO2 + eCO2 eNO3 eNO3 + eCO2 eNO2 + eNO3 eNO2 + eNO3 + eCO2 
Maple Atreatment 6.45bc 9.92a 6.37bc 9.05ab 6.23bc 8.11ab 5.18c 7.66ac 
year 1  (0.99) (0.88) (0.29) (2.27) (0.36) (0.67) (0.59) (1.39) 
 A760 8.45ab 9.92a 7.90ab 9.05ab 8.91ab 8.18ab 5.74b 7.66ab 
  (1.67) (0.88) (0.73) (2.27) (0.27) (0.67) (1.31) (1.39) 
 gs treatment 0.093a 0.066ab 0.086a 0.051bcd 0.065abc 0.035d 0.054abcd 0.044bcd 
  (0.022) (0.007) (0.011) (0.020) (0.010) (0.006) (0.011) (0.009) 
Maple Atreatment 4.45ab 4.95ab 3.88ab 3.63ab 3.60b 3.51b 4.22ab 5.07a 
year 2  (0.57) (1.10) (0.65) (0.18) (0.67) (0.54) (0.75) (0.93) 
 A760 6.59a 5.07bc 5.50ab 3.79cd 4.88bcd 3.58d 6.08ab 5.24ab 
  (0.61) (1.10) (0.25) (0.18) (0.49) (0543) (0.99) (0.93) 
 gs treatment 0.047ab 0.023c 0.046ab 0.016c 0.060ab 0.018c 0.071a 0.029bc 
  (0.009) (0.005) (0.007) (0.002) (0.026) (0.003) (0.018) (0.009) 

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 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).

Figure 2.

Total leaf area (cm2) and SLA (cm2 g1). 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 ha1 year1 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.

Figure 2.

Total leaf area (cm2) and SLA (cm2 g1). 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 ha1 year1 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.

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 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.

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).

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).

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 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 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.

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.

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.

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