Constraints to nitrogen acquisition of terrestrial plants under elevated CO2.

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Received Date : 16-Dec-2014 Accepted Date : 02-Mar-2015 Article type

: Primary Research Articles

Title: Constraints to Nitrogen Acquisition of Terrestrial Plants under Elevated CO2

Running head: Nitrogen acquisition under elevated CO2 Authors: Zhaozhong Feng1,8*, Tobias Rütting1,9, Håkan Pleijel1, Göran Wallin1, Peter B. Reich2,7, Claudia I. Kammann3, Paul C. D. Newton4, Kazuhiko Kobayashi5, Yunjian Luo6, Johan Uddling1* Affiliations: 1

Department of Biological and Environmental Sciences, University of Gothenburg, P.O. Box

461, 405 30 Gothenburg, Sweden. 2

Department of Forest Resources, University of Minnesota, 1530 Cleveland Avenue North,

St. Paul, MN 55108, USA. 3

Department of Plant Ecology, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 26-32,

35392 Giessen, Germany. 4

AgResearch Grasslands, Private Bag 11008, Palmerston North, New Zealand.

5

Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi,

Bunkyo-ku, Tokyo 113-8657, Japan. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/gcb.12938 This article is protected by copyright. All rights reserved.

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6

Institute of Urban Environment, Chinese Academy of Sciences, 361021, Xiamen, China.

7

Hawkesbury Institute for the Environment, University of Western Sydney, Penrith NSW

2753 Australia. 8

State Key Laboratory of Urban and Regional Ecology, Research Center for

Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 100085, Beijing, China. 9

Department of Earth Sciences, University of Gothenburg, P.O. Box 460, 405 30

Gothenburg, Sweden.

The correspondence authors are Zhaozhong Feng, Telephone:+86-10-62943823, Fax nunber: +86-10-62943822. E-mail: [email protected], and Dr. Johan Uddling, Telephone: +46-31 7866663, Fax number: +46-31 7862560, E-mail address: [email protected]

Keywords: carbon dioxide, crops, diversity, fertilization, FACE, forest, grassland, growth dilution, meta-analysis, nitrogen

The type of paper: Primary Research

Abstract A key part of the uncertainty in terrestrial feedbacks on climate change is related to how and to what extent nitrogen (N) availability constrains the stimulation of terrestrial This article is protected by copyright. All rights reserved.

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(1, 4, 9 and 16 species). Observations from different years were considered independent because the variance among years was similar in magnitude to that among experiments. However, the relationship between responses of ANPP and Nac to eCO2 was investigated also using data from multi-year experiments averaged across years, to ascertain the robustness of main conclusions.

Treatment of data

We conducted two main types of analyses: 1) investigation of the relationships between the effects of eCO2 on productivity (ANPP or NPP) and the corresponding Nac or N concentration, and if these differed among ecosystem types (grasslands, croplands, forests), plant functional types (C3 grasses, C4, non-legume forbs, legumes), N application rates, and levels of species diversity (1, 4, 9 and 16 species); 2) investigation of the development of effects of eCO2 on productivity and Nac over time in long-term (≥ 7 years) experiments. It is recognized that relationships between effects of eCO2 on productivity and Nac suffer from auto-correlation, since Nac is the product of the biomass produced and its N concentration (for forests also accounting for N resorption during senescence). Therefore, these relationships were compared with relationships between effects of eCO2 on productivity and N concentration (which do not suffer from auto-correlation) in order to substantiate interpretations of productivity–Nac relationships.

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INTRODUCTION

Terrestrial ecosystems are fertilized by rising atmospheric CO2 (Ainsworth & Long, 2005; de Graaff et al., 2006; Luo et al., 2006) and currently slow climate change by sequestering about one fourth of anthropogenic CO2 emissions (Le Quere et al., 2013). However, there is concern that this fraction will diminish in the future. The uncertainty regarding the impact of rising atmospheric CO2 on the magnitude of the terrestrial C sink is presently the largest unknown for terrestrial ecosystem feedbacks on climate change (IPCC, 2013). A key part of this uncertainty is related to how and to what extent nitrogen (N) availability constrains the elevated CO2 (eCO2)-induced stimulation of terrestrial net primary production – as often seen in field experiments (Schneider et al., 2004; Reich et al., 2006; Norby et al., 2010; Reich & Hobbie, 2013) – and whether or not this constraint will become stronger over time (Hungate et al., 2003; Luo et al., 2004). Plants growing in eCO2 often have increased N acquisition, but the relative increase in N acquisition is typically smaller than the relative stimulation of biomass production (Luo et al., 2006). Consequently, plant tissue N concentrations are usually decreased in eCO2, at both the leaf and whole-plant levels (Ainsworth & Long, 2005; Luo et al., 2006). At the leaf level, such decrease corresponds with metabolic down-regulation of the eCO2-induced stimulation of photosynthesis (e.g. Stitt & Krapp, 1999; Ellsworth et al., 2004). Furthermore, plant litter produced in eCO2 usually also has lower N concentration (Norby et al., 2001; for experiments included in this study, see Table S1), potentially making it less readily decomposed (Strain & Bazzaz, 1983; Luo et al., 2004), although effects on decomposition rates are highly variable and often small (Norby et al., 1999, 2001 and references therein). This article is protected by copyright. All rights reserved.

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Huang JY, Yang HJ, Yang LX et al. (2004) Effects of free-air CO2 enrichment (FACE) on yield formation of rice (Oryza sativa L.) and its interaction with nitrogen. Scientia Agricultura Sinica, 37, 1824–1830. (in Chinese) Hungate BA, Dukes JS, Shaw MR, Luo Y, Field CB (2003) Nitrogen and Climate Change. Science, 302, 1512–1513. Hungate BA, Stiling PD, Dijkstra P et al. (2004) CO2 elicits long-term decline in nitrogen fixation. Science, 304, 1291–1291. IPCC Climate Change (2013) The Physical Science Basis: Summary for Policymakers. (eds Stocker,T. et al.), Cambridge Univ. Press. Iversen CM (2010) Digging deeper: fine-root responses to rising atmospheric CO2 concentration in forested ecosystems. New Phytologist, 186, 346–357. Körner C (2006) Plant CO2 responses: An issue of definition, time and resource supply. New Phytologist, 172, 393–411. Kammann C, Grunhage L, Gruters U, Janze S, Jager HJ (2005) Response of aboveground grassland biomass and soil moisture to moderate long-term CO2 enrichment. Basic and Applied Ecology, 6, 351–365. Kim HY, Lieffering M, Kobayashi K, Okada M, Miura S (2003) Seasonal changes in the effects of elevated CO2 on rice at three levels of nitrogen supply: a free air CO2 enrichment (FACE) experiment. Global Change Biology, 9, 826–837. Kim HY, Lieffering M, Miura S, Kobayashi K, Okada M (2001) Growth and nitrogen uptake of CO2-enriched rice under field conditions. New Phytologist, 150, 223–229.


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Ko J, Ahuja L, Kimball B et al. (2010) Simulation of free air CO2 enriched wheat growth and interactions with water, nitrogen, and temperature. Agricultural and Forest Meteorology, 150, 1331–1346. Kongstad J, Schmidt IK, Riis-Nielsen T, Arndal MF, Mikkelsen TN, Beier C (2012) High resilience in heathland plants to changes in temperature, drought, and CO2 in Combination: Results from the CLIMAITE Experiment. Ecosystems, 15, 269–283. Kuzyakov Y (2002) Review: Factors affecting rhizosphere priming effects. Journal of Plant Nutrition and Soil Science, 165, 382–396. Larsen KS, Andresen LC, Beier C et al. (2011) Reduced N cycling in response to elevated CO2, warming, and drought in a Danish heathland: Synthesizing results of the CLIMAITE project after two years of treatments. Global Change Biology, 17, 1884–1899. Le Quere C, Andres RJ, Boden T et al. (2013) The global carbon budget 1959-2011. Earth System Science Data, 5, 165–185. Le Quere C, Raupach MR, Canadell JG et al. (2009) Trends in the sources and sinks of carbon dioxide. Nature Geoscience, 2, 831–836. Lee TD, Barrott SH, Reich PB (2011) Photosynthetic responses of 13 grassland species across 11 years of free-air CO2 enrichment is modest, consistent and independent of N supply. Global Change Biology, 17, 2893–2904. Leuzinger S, Luo YQ, Beier C, Dieleman W, Vicca S, Korner C (2011) Do global change experiments overestimate impacts on terrestrial ecosystems? Trends in Ecology and Evolution, 26, 236–241.


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Rütting T, Andresen LC (2015) Nitrogen cycle responses to elevated CO2 depend on ecosystem nutrient status. Nutrient Cycling in Agroecosystems, DOI: 10.1007/s10705-015-9683-8. Rütting T, Clough TJ, Mueller C, Lieffering M, Newton PCD (2010) Ten years of elevated atmospheric carbon dioxide alters soil nitrogen transformations in a sheep-grazed pasture. Global Change Biology, 16, 2530–2542. Schneider MK, Luscher A, Richter M et al. (2004) Ten years of free-air CO2 enrichment altered the mobilization of N from soil in Lolium perenne L. swards. Global Change Biology, 10, 1377–1388. Shimono H, Okada M, Yamakawa Y, Nakamura H, Kobayashi K, Hasegawa T (2008) Rice yield enhancement by elevated CO2 is reduced in cool weather. Global Change Biology, 14, 276–284. Shimono H, Okada M, Yamakawa Y, Nakamura H, Kobayashi K, Hasegawa T (2009) Genotypic variation in rice yield enhancement by elevated CO2 relates to growth before heading, and not to maturity group. Journal of Experimental Botany, 60, 523–532. Stitt M, Krapp A (1999) The interaction between elevated carbon dioxide and nitrogen nutrition: The physiological and molecular background. Plant Cell and Environment, 22, 583–621. Strain BR, Bazzaz FA (1983) Terrestrial plant communities. In: CO2 and Plants (ed. Lemon, ER). Westview, Boulder, Colo., pp. 177–222.


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available at that time only allowed for analysis of temporal response trends of up to five years (Körner, 2006). Today, several FACE experiments conducted in grasslands and forests have lasted for ten years or longer, offering an unprecedented opportunity to evaluate whether or not field observations support the PNL hypothesis at decennial time scale and across different terrestrial ecosystem types. To improve the understanding of how eCO2 affects stand-level plant N acquisition and its relationship with productivity, we synthesized responses to eCO2 of stand-level annual aboveground or whole-plant net primary production (ANPP or NPP) and corresponding plant N acquisition (Nac) and N concentration in FACE experiments conducted in grassland, cropland and forest ecosystems. By exploring the relationship between responses of productivity (X-axis) and corresponding Nac or plant N concentration (Y-axis) to eCO2, it is possible to quantify and separate productivity-mediated effects of eCO2 on Nac (slopes) from effects in the absence of eCO2-induced changes in productivity (intercepts; Pleijel & Uddling, 2012). This type of analysis thus allows for a functional interpretation of plant productivity, Nac and N concentration responses to eCO2 that differs from and complements the quantification of mean responses in traditional meta-analyses (Curtis, 1996; Ainsworth & Long, 2005; Luo et al., 2006). For example, it allows for a test of the hypothesis that decreased plant N concentration in eCO2 is linked to productivity enhancement, i.e. growth dilution. Furthermore, inclusion of all data available from large-sized FACE experiments allowed for comparisons of responses among different ecosystem types (grasslands, croplands and forests), plant functional types (C3 grasses, C4 grasses, legumes, non-legume forbs), and levels of species diversity or N fertilization. Finally, we analysed long-term


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previous year (Finzi et al., 2007; Norby et al., 2010). For grasslands and croplands, annual Nac was calculated as the N content of biomass produced in that year, without accounting for within-plant N recycling in perennial grassland species because available data did not allow for such estimation. Relative effects of eCO2 on grassland Nac reported here would be unrepresentative for effects on N uptake (i.e. the N required to produce the biomass in that year) if N resorption efficiency was significantly affected by CO2 treatment. However, there were no effects of eCO2 on leaf N resorption efficiency in past studies in FACE experiments (e.g., Finzi et al., 2001; Lindroth et al., 2001; Norby & Iversen, 2006; Esmeijer-Liu et al., 2009) or in a review of earlier experiments (Norby et al., 2001). Mini-FACE experiments (1-4 m in diameter) were excluded from the present study out of concern for potential large edge effects associated with the small CO2 enrichment footprint. Main focus had to be on aboveground responses as studies in grasslands and croplands usually did not report root data. However, for the six out of 13 experiments reporting both aboveground and belowground data, analyses were conducted also on whole-plant NPP and Nac. Data were compiled from 35 peer-reviewed articles and two open-source web sites (Table 2). Mean, standard deviation, and replication (n) of plots in ambient (aCO2) and elevated (eCO2, ~450-600 ppm) CO2 concentration were taken from tables, digitized from figures, or directly obtained from the authors of the papers. Observations were considered independent if they were made on different plant communities, in different years, and at different levels of additional treatments, e.g. N fertilization. However, BioCON data were averaged across different community compositions at each of four levels of species diversity


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(1, 4, 9 and 16 species). Observations from different years were considered independent because the variance among years was similar in magnitude to that among experiments. However, the relationship between responses of ANPP and Nac to eCO2 was investigated also using data from multi-year experiments averaged across years, to ascertain the robustness of main conclusions.

Treatment of data

We conducted two main types of analyses: 1) investigation of the relationships between the effects of eCO2 on productivity (ANPP or NPP) and the corresponding Nac or N concentration, and if these differed among ecosystem types (grasslands, croplands, forests), plant functional types (C3 grasses, C4, non-legume forbs, legumes), N application rates, and levels of species diversity (1, 4, 9 and 16 species); 2) investigation of the development of effects of eCO2 on productivity and Nac over time in long-term (≥ 7 years) experiments. It is recognized that relationships between effects of eCO2 on productivity and Nac suffer from auto-correlation, since Nac is the product of the biomass produced and its N concentration (for forests also accounting for N resorption during senescence). Therefore, these relationships were compared with relationships between effects of eCO2 on productivity and N concentration (which do not suffer from auto-correlation) in order to substantiate interpretations of productivity–Nac relationships.


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In the first type of analysis, relative effects (% change in eCO2 compared to aCO2) on Nac or N concentration (Y-axis) were plotted against the corresponding relative effects on ANPP or NPP (X-axis). Since the X-axis variable is measured with error and since the direction of causality between X- and Y-variables likely works in both ways, reduced major axis (RMA) regression rather than ordinary least squares (OLS) regression was used to determine the best fit for a linear bivariate relationship (Falster et al., 2006). Homogeneity of Y-intercepts and slopes with respect to group (ecosystem type, plant functional type, level of species diversity or N supply) were tested by analysis of co-variance using the software SMATR (Falster et al., 2006). A significant difference in Y-intercept among groups can be readily interpreted only if the slopes do not significantly differ among groups. Effects were considered statistically significant at P ≤ 0.050. The slope of the productivity–Nac relationship reveals if a certain change in the productivity response to eCO2 is accompanied by an equal relative change in Nac (slope = 1), or if Nac responses are proportionally greater (slope > 1) or less (0 < slope < 1; i.e. growth dilution) than changes in productivity. The Y-intercept of the relationship demonstrates if eCO2 has any effect on Nac in addition to those mediated by changes in productivity. The relationship between responses of productivity (X-axis) and N concentration (Y-axis) reveals if a possible decrease in N concentration in eCO2 is linked to growth dilution. If so, the relationship should pass through the origin and have a negative slope. Comparisons of plant functional types and levels of species diversity were made using BioCON data only. In the comparison of plant functional types, data were averaged such that each functional type contributed 52 data points (13 years × 2 N levels × 2 levels of species


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diversity (1 and 4)). In the comparison of species diversity levels, averaging was made as described for the entire dataset above, with each diversity level contributing 26 data points (13 years × 2 levels of N). To investigate the long-term dependence of responses to eCO2 on the duration of CO2 exposure, only perennial vegetation which was exposed ≥ 7 years was included. The statistical significance of temporal trends across all long-term studies was based on t-tests of all individual temporal response trends (n = 8). The overall mean response to eCO2 at each exposure year across all long-term FACE experiments and at each site across multiple years was calculated by MetaWin 2.1(Rosenberg et al., 2000). A mixed-effect model was used, assuming random variation in effect size (the ratio of the effect variable between eCO2 and aCO2) among FACE studies. The analysis used the mean, standard deviation and n in both aCO2 and eCO2.

RESULTS

Across all three ecosystem types, eCO2 affected Nac in two ways (Fig. 1a): by a positive ANPP-mediated effect (slope = 1.15 ± 0.08; P < 0.001; r2 = 0.68) and by a negative effect at modest (slight positive or negative, or neutral) impact on ANPP (intercept = –10.3%; P < 0.001). The slopes did not significantly differ among ecosystem types (i.e., no significant ANPP × Ecosystem interaction), while the negative intercepts were close to being significant among the three ecosystem types (P = 0.053; they were –9 ± 2% in grasslands, –14 ± 3% in croplands and –15 ± 8% in forests; ± 95% confidence interval). Across all data,


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eCO2 increased ANPP by 16% (± 2%) and Nac by 8% (± 2%). Although productivity and Nac were overall positively affected by eCO2, both modest productivity responses (±10 % or smaller changes) and negative Nac responses to eCO2 were rather common phenomena, each accounting for a third of the data points in Fig. 1a. The N concentration of aboveground annual biomass production was decreased by eCO2 by 6% (± 2%), 8% (± 2%), and 12% (± 2%) in grassland, cropland and forest ecosystems, respectively, and this effect was not related to productivity enhancement (Fig. 1b). None of these results (slopes or intercepts) are consistent with the growth dilution hypothesis. Across the seven FACE experiments with factorial CO2 × N treatments, N fertilization had similarly strong positive effects on ANPP and Nac in ambient and elevated CO2 (Fig. 2). This was the case also for a subset of data for which the effects of eCO2 on ANPP ranged from -10% to +10% in the unfertilized or low N treatment. Large positive effects of independent N additions on Nac indicate strong plant N demand in these ecosystems, under ambient as well as elevated CO2. These results strongly suggest that negative effects of eCO2 on Nac at neutral or modest shifts in productivity were not associated with decreased plant N demand. The relationship between responses of ANPP and Nac to eCO2 based on data from multi-year experiments averaged across years confirmed the negative effect on Nac at unchanged productivity (i.e., intercept –10 ± 3%) and the lack of growth dilution (i.e., the slope was not < 1; Fig. 3). This result demonstrated that the conclusions drawn did not depend on the treatment of data from different years as independent observations.


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Categorizing data into groups with very high (> 25 g N m-2 yr-1), high (12.6 - 25.0 g N m-2 yr-1) and intermediate or no (0 - 12.5 g N m-2 yr-1) external N supply, it was found that the negative effect of eCO2 on Nac at unchanged productivity was strongest at highest N supply (P < 0.001; Fig. 4). This result demonstrated that this negative effect eCO2 on Nac cannot be alleviated by increasing N fertilization. The negative effect of eCO2 on Nac at unchanged productivity observed in aboveground plant parts (Fig. 1a) was present also at the whole-plant level (i.e., for whole-plant NPP and Nac data the intercept was -10%; Fig. 5a) when analysing data for the six out of 13 experiments where root biomass data were available. The whole-plant N concentration was decreased by 5% (± 1%), 8% (± 1%) and 6% (± 4%) in grassland, cropland and forest ecosystems, respectively, and this effect was not coupled to productivity enhancement (Fig. 5b). Root N concentration was on average decreased by eCO2 (–4%), albeit to a lesser extent than aboveground plant N concentration (–7%; Fig. S1). These findings strongly indicate that the results and conclusions based on aboveground data from all 13 experiments hold at the whole-plant level. In trees, the relative distribution of biomass or N to leaf versus wood production was similar in aCO2 and eCO2, and plant N concentrations were similarly decreased by eCO2 in wood, leaves and litter (Fig. 6a-b). Moreover, foliage, the largest component of tree aboveground Nac, exhibited decreased total N content at eCO2 when there was no effect of CO2 treatment on total leaf biomass production (-14%; Fig. 6c), similarly to the patterns for aboveground and whole-plant data (Fig. 1a, Fig. 5a). This analysis of forest data shows that the eCO2-induced decrease in Nac at unchanged productivity was not related to shifts in


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within-plant C or N allocation to organs with different C:N ratios (i.e., wood versus leaves) or to eCO2-induced effects on N resorption efficiency. In a separate analysis of data from the BioCON grassland experiment, it was found that the negative effect of eCO2 on Nac at unchanged productivity was different in magnitude and significance among plant functional types. The negative effect was significant in C3 grasses (–10 ± 4%; mean ± 95% confidence interval) and non-legume forbs (–14 ± 6%) but not in C4 grasses (–4 ± 7%) or legumes (–2 ± 8%; Table 3). The slope of the relationship between responses of productivity and Nac to eCO2 was lower in non-legume forbs than in the other plant functional types. In an analysis categorizing BioCON data according to species diversity, neither intercepts nor slopes of the relationships between responses of productivity and Nac to eCO2 differed among plant communities with 1, 4, 9 or 16 species (Table 3). These results demonstrate that the negative effect of eCO2 on Nac at neutral or modest shifts in productivity was restricted to non-legume C3 plants; however, it was present at different levels of species diversity (all functional types were present at all species diversity levels in this analysis). The responses of ANPP (P = 0.98) and Nac (P = 0.18) did not, on average, decline over time across all long-term (7-11 years) FACE experiments (Fig. 7); instead there was a significant trend that eCO2-induced decreases in plant N concentration diminished over time (P = 0.026). Temporal response trends were similar for aboveground and whole-plant data in experiments reporting root data (cf. Fig. 8 and Fig. S2). Looking at response trends of individual experiments, stimulation of productivity and Nac to eCO2 decreased over time in one of the long-term forest experiments (Norby et al., 2010); however, constant or increasing


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productivity and Nac responses over time were more common in the entire dataset (Fig. 8). The declining response trend observed in the sweetgum (Liquidambar styraciflua L) plantation of the Oak Ridge experiment contrasts with observations of sustained productivity enhancements over a decade in forest experiments with loblolly pine (Pinus taeda ) in the Duke FACE experiment or northern hardwood species in the Aspen FACE experiment. Data from the latter experiment (Talhelm et al., 2014), as well as from the last few years of the Duke FACE experiment, were not included in the present long-term analysis since long-term data on annual Nac were not available. In grasslands, the ecosystem type from which most of the long-term data were derived, temporal response trends did not consistently vary with level of N application (Fig. 8) and were similar at low and high species diversity (Fig. S3). Taken together, these results on long-term temporal response trends show that N limitations constraining productivity responses to eCO2 typically did not progress at decennial time scale.

DISCUSSION

This synthesis revealed a relationship between responses of productivity and Nac to eCO2 that was positive and strong and exhibited a negative offset, such that eCO2 had a negative effect on Nac (–10% to –12%) at modest or neutral shifts in productivity (Figs. 1a, 5a). The positive slope of the relationship was expected and likely reflects growth-mediated enhanced nutrient foraging by plants with more extensive and/or deeper root systems and larger whole-plant N sink strength. The findings that plants in eCO2 often take up more N and that large growth stimulation by eCO2 is accompanied by enhanced N acquisition have been emphasized in This article is protected by copyright. All rights reserved.

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previous studies (Norby et al., 1999; Luo et al., 2006; Finzi et al., 2007; Iversen, 2010) and will not be discussed further here. Instead, we will focus on the finding of a negative effect of eCO2 on plant Nac and N concentration at neutral or modest shifts in productivity. This effect was found in all three ecosystem types (Figs. 1, 5) and regardless of the levels of external N supply (Fig. 4) or species diversity (Table 3). It was found to be similar for aboveground and whole-plant data (cf. Fig 1 and Fig. 5) and regardless if data from individual years were treated as independent or not (cf. Fig 1a and Fig. 3). For forests, the effect was not related to eCO2-induced effects on within-plant C or N allocation or N resorption efficiency (Fig. 6). The negative effect of eCO2 on plant Nac and N concentration at neutral or modest shifts in productivity has two important implications. First, it refutes the growth dilution hypothesis, which attributes the decrease in N concentration to N uptake not keeping pace with productivity stimulation (Poorter et al., 1997; Gifford et al., 2000; Loladze, 2002; Taub & Wang, 2008). In fact, the decrease in N concentration under eCO2 was totally independent of the presence or magnitude of eCO2-induced productivity enhancement (Figs. 1b & 5b). Second, the negative effect of eCO2 on Nac at unchanged productivity indicates that when eCO2 had small or no effects on productivity, the potential positive effect of increased substrate availability for photosynthesis and growth was balanced out by a negative effect on plant N acquisition. It may be argued that the negative effect of eCO2 on Nac at unchanged productivity was a consequence of decreased Rubsico demand translating into decreased whole-plant N demand in eCO2 (hypothesis N4 in Table 1). It is indeed likely that much, or perhaps even most, of the decrease in Nac at unchanged productivity was accounted for by decreased foliar


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Rubisco content, as indicated by an earlier FACE meta-analysis of leaf-level responses (Long et al., 2004). However, such decrease in Rubisco might not indicate decreased whole-plant N demand under eCO2, since N fertilization had similarly strong positive effects on ANPP and Nac in both ambient and elevated CO2 across the seven FACE experiments with factorial CO2 × N treatments (Fig.2). Productivity was markedly N limited also at other FACE sites, likely in both ambient and elevated CO2 (Oren et al., 2001; Norby et al., 2010). These results indicate that decreased leaf Rubsico content should be viewed as a mechanism by which plants allocate a given amount of N more efficiently under eCO2 (i.e. less to Rubisco, more to foliage production; McMurtrie et al., 2008) rather than as a response that decreases plant N demand at the stand level. Plants with minimal productivity responses to eCO2 thus likely acquired less N than ambient CO2-grown counterparts because access was somehow decreased, and not because demand was lower. This implies that decreased Nac was likely at least part of the reason for, and not the consequence of, the numerous observations of absent or modest productivity responses to eCO2 (Figs. 1 & 5). The present study is, to our knowledge, the first to indicate such causal link between stand-level inhibition of Nac and lack of productivity responses to eCO2 across different types of ecosystems. The negative effect of eCO2 on Nac at unchanged productivity cannot be attributed to one particular mechanism or process by which eCO2 influences Nac (Table 1), since none of them is entirely consistent with observations. The effect was not a result of decreased Rubisco content lowering whole-plant N demand (hypothesis N4 in Table 1), as discussed above. The ‘net immobilization’ hypothesis (N2 in Table 1) is in conflict with a recent FACE meta-analysis showing that gross (microbial) N immobilization and gross N mineralization


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were stimulated to the same extent by eCO2 (Rütting & Andresen, 2015). This hypothesis is also counter to observations that soil mineral N content was typically not significantly affected by eCO2 in the grassland experiments of the present study (Gloser et al., 2000; Newton et al., 2010; Larsen et al., 2011). Moreover, the net immobilization hypothesis as well as the hypothesis of slower decomposition of litter with lower N concentration in eCO2 leading to PNL (N1 in Table 1) also fails to explain why the negative effect of eCO2 on Nac at unchanged productivity was stronger at higher N supply (Fig. 4) and was found also in experiments with fertilized annual crops (Fig. 1), or why the eCO2-induced effects on productivity or Nac typically did not decline over time (Figs. 7 & 8). The hypothesis of transpiration-mediated negative effects on plant N concentration under eCO2 (N5 in Table 1) is in conflict with the finding that stomatal conductance was equally decreased by eCO2 in C3 and C4 plants (Lee et al., 2011) while the negative effect on Nac at unchanged productivity was observed in C3 plants but not in C4 plants in the BioCON experiment (Table 3). Finally, the inhibited shoot nitrate assimilation hypothesis (N3 in Table 1) fails to explain why the eCO2-induced decrease in plant N concentration diminished over time (Fig. 7). Different mechanisms and processes by which eCO2 negatively affects plant Nac act in parallel and their relative importance likely differ among ecosystems. The finding that the negative effect of eCO2 on Nac at neutral or modest shifts in productivity was significant in non-legume C3 plants but not in C4 plants in the BioCON experiment (Table 3) indicates that inhibited shoot nitrate assimilation in C3 (but not in C4) plants under eCO2 (hypothesis N3 in Table 1) may play an important role (Bloom et al., 2010, 2012). This hypothesis also agrees with the results indicating that the effect was not associated with effects of eCO2 on net


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immobilization (the effect was present in fertilized plants and annual crops; Figs. 1 and 4) or decreased whole-plan N demand (Fig. 2). However, elucidation of the potential roles played by different candidate mechanisms and processes requires experiments and measurements designed to separate the influences of total availability of soil N from the influence of soil N form (nitrate, ammonium, organic) on plant N acquisition under eCO2. The lack of a negative effect of eCO2 on Nac at neutral or modest shifts in productivity in legumes (Table 3) was expected. This is likely a result of their access to N through N-fixing symbiotic bacteria, making them less dependent on both soil N availability and shoot nitrate assimilation. The expectation that the magnitude of positive ecosystem productivity responses to eCO2 will decline over time due to PNL (Luo et al., 2004; Körner, 2006; Leuzinger et al., 2011) was not supported by our analysis of long-term (7-11 years) responses of plant productivity, Nac and N concentration in FACE experiments (Figs. 7, 8 & Fig. S2). It is unclear why declining response trends were found in the sweetgum plantation of the Oak Ridge FACE experiment (Norby et al., 2010) but not in the Duke FACE experiment with loblolly pine (Duke FACE; McCarthy et al., 2010). It has been speculated that greater nutrient demand of the deciduous sweetgum trees compared with the evergreen pine trees may have caused N deficiency and associated growth decline to occur earlier in the Oak Ridge FACE experiment than in the Duke FACE experiment (Norby et al., 2010). In a third long-term FACE experiment—not included in the present analysis of temporal trends due to lack of long-term Nac response data—aggrading stands of northern hardwood species exhibited decennial sustained productivity enhancement in eCO2 (Aspen FACE, Talhelm et


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al., 2014.); however, expanding young tree stands are not ideal to assess the PNL hypothesis (Körner 2006). Additional long-term forest FACE experiments are critically needed to understand where and when to expect PNL of eCO2-induced forest productivity, and where and when not to. The temporal stability of ecosystem productivity responses observed here (Fig.7) is consistent with the increased global net CO2 uptake by land and oceans that has persisted during the past 50 years (Le Quere et al., 2009; Ballantyne et al., 2012). While the positive effect of species diversity per se on productivity and Nac increased over time in the BioCON experiment (Reich et al., 2012), there was no indication of long-term responses to eCO2 to vary with species diversity in this meta-analysis (Fig. S3). Neither did the level of N application systematically affect temporal response trends (Fig. 8), which does not support expectations based on the PNL hypothesis (Luo et al., 2004). While the relationship between responses of productivity and Nac to eCO2 was similar in grassland, cropland and forest ecosystems (Fig. 1a), the implications are partly ecosystem type-specific. For grasslands, predictions of plant productivity and N concentration responses to eCO2 are difficult to make, since species belonging to different functional types behave differently (Table 3). Indirect effects of eCO2 on community composition could thus potentially dominate these responses in grassland ecosystems, but such effects are modest in some experiments (Reich & Hobbie, 2013) and more important in others (Grüters et al., 2006; Newton et al., 2014). It is also possible that because many grasslands are comprised of mixed functional groups, their response to eCO2 will be stabilized by high functional diversity (even if individual species responses are not) as is true more generally (Gross et al., 2014). For croplands, it was demonstrated that eCO2 decreases crop quality and that this


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effect is independent of the presence or magnitude of yield stimulation (Fig. 1b) and cannot be alleviated by increasing N fertilization (Fig. 4). Our study included experiments with both wheat and rice, the two crop species contributing most to human caloric intake, and thus has important implications for human nutrition and global food security (Myers et al., 2014). These results are in line with recent studies reporting on decreased grain N concentration (Pleijel & Uddling, 2012) and inhibited nitrate assimilation (Bloom et al., 2014) in wheat under eCO2. For forests, the results indicate that the terrestrial carbon sink (which is dominated by forests) is indeed constrained by N limitations, with neutral or modest shifts in productivity being associated with negative effects of eCO2 on Nac (Fig. 1a). However, data from additional FACE forest experiments are required to better understand the processes governing the long-term responses of forest productivity to eCO2, with declining response trends of productivity and Nac being found in one out of three of the long-term FACE experiments to date (McCarthy et al., 2010; Norby et al., 2010; Talhelm et al., 2014). The results of this study have important implications for our understanding of, and attempts to model the future global C cycle. First, and most importantly, we found that eCO2 decreased plant N acquisition at neutral or modest shift in productivity and that the decrease in plant N concentration is independent of the presence or magnitude of eCO2-induced productivity enhancement. These effects, which were found in all three ecosystem types and regardless of levels of N supply or species diversity, were likely linked to decreased plant N access rather than to decreased plant N demand. Second, averaging data across all long-term FACE experiments, we found no evidence of PNL constraining the eCO2-induced productivity enhancement (Fig. 7) at decennial time scale. Both these findings contrast with


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the representation of N limitations of plant productivity responses to eCO2 in terrestrial ecosystem models (Parton et al., 2010; Zaehle et al., 2014), which presume that the decrease in plant N concentration in eCO2 is an indirect consequence of increased biomass production; occurring through growth dilution and, over time, PNL. Taken together, our results suggest that in addition to low soil fertility acting to constrain the eCO2 fertilization effect, N constraints of eCO2-induced terrestrial productivity enhancement acting at decennial time scale are likely the result of negative effects of eCO2 on plant N acquisition that are independent of the processes that may lead to progressive N limitation.

Acknowledgements We are grateful to the authors of the studies whose valuable field work provided the data for this meta-analysis. The study was financially supported by the strategic research area Biodiversity and Ecosystem services in a Changing Climate (BECC, www.cec.lu.se/research/becc), Sweden. The first and last authors were also supported by the Hundred Talents Programme of Chinese Academy of Sciences and the Swedish Research Council Formas (214-2010-382), respectively. BioCON research has been supported by the Department of Energy (DOE/DE-FG02-96ER62291) and the National Science Foundation (NSF Biocomplexity 0322057, NSF LTER DEB 9411972 (1994–2000), DEB 0080382 (2000–2006), and DEB 0620652 (2006–2012), and NSF LTREB 0716587).


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Supporting information captions Table S1. Aboveground litter N concentration (%) across species, N levels and experimental years at each FACE site Fig. S1. Effect of elevated CO2 on the N concentration ([N]) of the annual production of above- and belowground plant parts. Fig. S2. Effect of elevated CO2 (eCO2) on (a) whole-plant net primary production (NPP) and (b) corresponding nitrogen acquisition (Nac) in relation to the number of years of experimental CO2 exposure at each FACE site. This article is protected by copyright. All rights reserved.

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Fig. S3. Effect of elevated CO2 (eCO2) on (a) aboveground net primary production (ANPP) and (b) corresponding nitrogen acquisition (Nac) in relation to the number of years of experimental CO2 exposure in grassland ecosystems with low diversity and high diversity.

Table 1. Hypothesized mechanisms and processes by which eCO2 may negatively (#N) or positively (#P) affect plant N acquisition. Papers cited discuss, but do not necessarily endorse, the hypotheses under question. #

Hypothesis

Selected references

#N1

Slower decomposition of litter with lower N concentration decreasing soil N availability over time

Rastetter et al. 1992; Luo et al. 2004

#N2

Net N immobilization by a larger soil microbial biomass

Diaz et al. 1993; de Graaff et al. 2006; Hu et al. 2006

#N3

Inhibited shoot nitrate assimilation in C3 plants

Bloom et al. 2010, 2012

#N4

Decreased Rubisco demand

Long & Drake 1992; Long et al. 2004; McMurtrie et al. 2008

#N5

Decreased transpiration-driven mass-flow of soil N to roots

McDonald et al. 2002; McGrath & Lobell 2013

#P1

Enhanced foraging by plants with larger/deeper root systems and larger whole-plant N sink strength

Finzi et al. 2007

#P2

Stimulated decomposition and N mineralization by a larger and/or more active microbial community (so called ‘priming’)

Kuzyakov 2002; Dijkstra et al. 2008; Rütting et al. 2010; Phillips et al. 2011

#P3

Increased N2-fixation and, over time, N availability in

Poorter et al. 1997; Zak et al.

ecosystems with N-fixing taxa

1993; Watanabe et al. 2013


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Table 2. Description of the large-scale Free-Air CO2 Enrichment (FACE) experiments in grassland, cropland and forest ecosystems included in the present meta-analysis. Name

Locatio n

Start Expo sure

Cedar Creek, MN,US A

Longit ude Latitu de 93°12 ’W 45°24 ’N

BioCO N

Swiss-F ACE

Eschiko n, Switzerl and

8°41’ E 47°27 ’N

NZ-FA CE

Bulls, New Zealand

GiFAC E

Data availa ble

Other treatments

Ecosystem, species

Referen ce

Low N, 0 g m-2 yr-1 High N, 4 g m-2 yr-1 Diversity levels (1, 4, 9, 16 species) Low N, 10 g m-2 yr-1 (1993), 14 g m-2 yr-1 (1994-2002) High N, 42 g m-2 yr-1(1993), 56 g m-2 yr-1 (1994-2002) Low frequency cut, 4 times per year High frequency cut, 7 (1993) and 8 times per year (1994-2002)

Managed grassland C3, C4 grasses, legumes and forbs

Reich et al. 2001, 2004, 2006; Reich 2009

Managed grassland Monocultur e (Lolium perenne) Biculture (L. perenne and Trifolium repens)

Hebeise n et al. 1997; Schneid er et al. 2004; Zanetti et al. 1997

Sheep grazed pasture C3 and C4 grasses, legumes and forbs Am N addition: 4 g Semi-natur al meadow bien m-2 yr-1 Dominated t

Newton et al. 2010; Ross et al. 2004

Can opy clos ure 1998-2 010

eC O2 (pp m) 550

1993

1993-2 002

600

175°1 6’E 40°14 ’S

1997

1997-2 007

475

Giessen, 8°41’ German E y 50°32

1998

1998-2 008

1998


Kamma nn et al. 2005*

Accepted Article

’N

+20 %

CLIMA ITE

Copenh agen, Denmar k

11°58 ’E 55°53 ’N

2005

2007

China-F ACE

Wuxi, China

120°3 0’E 31°35 ’N

2002

2002-2 003

2001-2 003

2005 Yangzh ou, China

119°4 2’E 32°35 ’N

2005

2005-2 006

-

510

Summer drought, passive warming, or both

Am Low N, 12.5 g bien m-2 yr-1 (2002), 9 g t m-2 yr-1 +20 (2003) 0 Med N, 18 g m-2 yr-1 High N, 25 g Am m-2 yr-1 bien Low N, 15 g t +20 m-2 yr-1 Med N, 25 g 0 m-2 yr-1 Am High N, 35 g bien m-2 yr-1 t +20 Low N, 15 g m-2 yr-1 0 Am High N, 25 g bien m-2 yr-1 t +20 Low N, 12.5 g m-2 yr-1 0 High N, 25 g m-2 yr-1


by Arrhenathe rum elatius, Holcus lanatus, Alopecurus pratensis and Poa pratensis Semi-natur al heathland Dominated by Calluna vulgaris and Deschamps ia flexuosa Triticum aestivum

Oryza sativa

Triticum aestivum

Oryza sativa

Kongsta d et al. 2012; Larsen et al. 2011

Dong et al. 2002; Huang et al. 2004; Yang et al. 2006, 2007a,b, c, 2009,

Accepted Article

Rice-FA Iwate, CE Japan

140°5 7’E 39°38 ’N

1998

1998-2 000

2003-2 004

AZ-FA CE

Maricop a, Arizona , USA

111°5 9’W 33°4’ N

1992

1992-1 994

1994-1 996

1997-1 999

Braunsc hweig-F ACE

Braunsc hweig, German y

10°26 ’E 52°18 ’N

1999

1999-2 005

Am bien t +20 0

Low N, 4 g m-2 yr-1(1998) Med N, 8 g m-2 yr-1 (1998), 9 -2 -1 g m yr (1999-2000) Am High N, 12 g bien m-2 yr-1(1998), 15 g m-2 yr-1 t +20 (1999-2000) Four 0 genotypes Am Wet, ample bien water t Dry, 1/2 wet +20 treatment 0 Am Low N, 7 g bien m-2 yr-1(1996), 13.5 g m-2 t +20 yr-1(1997) High N, 35 g 0 m-2 yr-1 Am bien t +20 0 550

Oryza sativa

Oryza sativa

Triticum aestivum

Triticum aestivum

Adamse n et al. 2005; Ko et al. 2010

Sorghum bicolor

Wet, ample water Dry, 1/3 wet treatment Low N, 20.4 g m-2 yr-1(2000) High N, 46.9 g m-2 yr-1(2000);

Hordeum vulgare+ Lolium multiflorum Lam.

Low N, 6.3 g m-2 yr-1(2001) High N, 12.6 g Beta m-2 yr-1(2001); vulgaris


Kim et al. 2001, 2003; Shimon o et al. 2008, 2009

Weigel & Manders cheid, 2012

Accepted Article

Low N, 11.4 g m-2 yr-1(2002) High N, 25.1 g m-2 yr-1(2002); Triticum aestivum Low N, 14.1 g m-2 yr-1(2003) High N, 25.1 g m-2 yr-1(2003); Hordeum Low N, 7.8 g vulgare+ m-2 yr-1(2004) Lolium High N, 15.6 g multiflorum m-2 yr-1(2004); Lam. Low N, 8.4 g m-2 yr-1(2005) Beta High N, 16.8 g vulgaris m-2 yr-1(2005)

Triticum aestivum Duke FACE

ORNL FACE

Aspen FACE

Durham , NC,US A Oak Ridge, TN, USA

79°05 ’W 35°58 ’N 84°20 ’W 35°54 ’N

1997

1997-2 199 003 7

1998

1998-2 199 008 8

Rhinela nder, WI, USA

89°42 ’W 45°36 ’N

1998

2001-2 200 003 2 200 1

Am bien t+2 00 565

Pinus taeda Liquidamb ar styraciflua Liquidamb ar styraciflua

560

Populus tremuloides P. tremuloides & Betula papyrifera


Finzi et al. 2007

Finzi et al. 2007; Norby 2009; Norby et al. 2010 Finzi et al. 2007

Accepted Article

POP-FA Tuscany 11°48 CE , ’E Italy 42°22 ’N

1999

2000 2001

200 0

550

P. alba, P. nigra, P. euramerica na

* including unpublished data

Table 3. Relationships between responses of annual aboveground net primary production (ANPP; X axis) and corresponding nitrogen acquisition (Nac; Y axis) to elevated CO2 of different plant functional types (PFTs) and of different levels of species diversity (Div) in the BioCON experiment. P values for PFT could not be interpreted as the ANPP × PFT interaction was significant. Different letters indicates significant differences in slopes among PFTs or Div.

Contras t

Equation

P values

Slope

Intercept

ANPP ×

(±

(±

PFT or r2

n

ANPP

(PFT or Div

95%CI)

95%CI)

0.97

-7.8

All

Div)

0.83 (0.05)

(3.4)

C3

0.85

-10.3

0.

grasses

(0.10) a

(4.3)

82

C4

0.99

-3.62

PFTs

0.86 grasses

(0.10) a

208

< 0.001

52

< 0.001

52

< 0.001

(7.1)


0.016

-

Finzi et al. 2007

Accepted Article

1.02

-2.16

(0.11) a

(8.4)

Non-legu

0.79

-14.0

me forbs

(0.10) b

(5.8)

1.16

-9.7

Legumes

All (0.10)

(2.7)

0.97

-6.6

1 Div

(0.15)

(4.2)

(numbe

1.14

-9.0

4 r of

(0.21)

(7.6)

species)

1.27

-12.1

(0.22)

(6.7)

1.23

-10.6

(0.22)

(4.5)

9

16

0.85

52

< 0.001

0.77

52

< 0.001

0.80

104

< 0.001

0.83

26

< 0.001

0.76

26

< 0.001

0.79

26

< 0.001

0.78

26

< 0.001


0.13

0.99

Accepted Article

Figure legends Figure 1. Relationship between effects of elevated CO2 (eCO2) on annual aboveground net primary production (ANPP) and corresponding (a) nitrogen acquisition (Nac) or (b) nitrogen concentration ([N]) for grassland, cropland and forest ecosystems. The solid and dashed lines in (a) represent the regression and the 1:1 line, respectively. The fitted regression equation is Y = 1.15 (± 0.08) X – 10.3 (± 2.0) where the values within brackets represent 95% confidence intervals; r2 = 0.68 and n = 242.

Fig. 2. Effects of nitrogen (N) fertilization on annual aboveground net primary production (ANPP) and corresponding N acquisition (Nac) under ambient (aCO2) and elevated (eCO2) CO2. Data are from seven factorial CO2 × N FACE experiments conducted in grassland and cropland ecosystems (BioCON, Swiss-FACE, GiFACE, China-FACE, Rice-FACE, AZ-FACE, Braunschweig-FACE, see Table 2). (a) all data available (n = 264); (b) data for which effects of eCO2 on ANPP ranged from -10% to +10% in the unfertilized or low N treatment (n = 85). Error bars represent 95% confidence intervals.

Figure 3. Relationship between effects of elevated CO2 (eCO2) on aboveground net primary production (ANPP) and corresponding nitrogen acquisition (Nac) for grassland, cropland and forest ecosystems. For grasslands and forests, which were multi-year experiments, each data point represents the overall mean effect across years, calculated using MetaWin 2.1. The


Accepted Article

solid and dashed lines in (a) represent the regression and the 1:1 line, respectively. The fitted regression equation is Y = 1.02 (± 0.15) X – 9.7 (± 3.1) where the values within brackets represent 95% confidence intervals; r2 = 0.53 and n = 74. .

Figure 4. Relationship between effects of elevated CO2 (eCO2) on annual aboveground net primary production (ANPP) and corresponding nitrogen acquisition (Nac) for grassland and cropland ecosystems with different levels of N supply. The solid and dashed lines are the regressions for different levels of N supply and the 1:1 line, respectively. Equations (with values within brackets representing 95% confidence intervals) and values of r2 and n for each N supply level are given below: 0-12.5 g N m-2 yr-1: Y = 1.16 (± 0.09) X – 8.0(± 2.2); r2 = 0.73 and n = 157 12.5-25 g N m-2 yr-1: Y = 1.51 (± 0.31) X – 14.7 (± 5.0); r2 = 0.60 and n = 33 ≥ 25 g N m-2 yr-1: Y = 1.30 (± 0.37) X – 18.7 (± 8.2); r2 = 0.45 and n = 23

Figure 5. Relationship between effects of elevated CO2 (eCO2) on whole-plant net primary production (NPP) and corresponding (a) nitrogen acquisition (Nac) or (b) nitrogen concentration ([N]) for grassland, cropland and forest ecosystems. Data in this figure originate from the 6 experiments where both aboveground and belowground data were available (BioCON, Rice-FACE, Duke FACE, ORNL FACE, Aspen FACE, POP-FACE; see Table 2). The solid and dashed lines in (a) are the regression for different ecosystem types


Accepted Article

and the 1:1 line, respectively. Ecosystem type-specific equations (with values within brackets representing 95% confidence intervals) and values of r2 and n are given below: Grassland: Y = 1.22 (± 0.11) X – 9.96 (± 2.7) r2 = 0.77 and n = 104 Cropland: Y = 1.08 (± 0.30) X – 10.6 (± 5.2), r2 = 0.89 and n = 8 Forest: Y = 1.16 (± 0.30) X – 12.5 (± 12.6), r2 = 0.43 and n = 29

Figure 6. Effects of elevated CO2 (eCO2) on (a) the nitrogen (N) concentrations of wood, leaf and litter, and (b) on relative distribution of biomass or N acquisition (Nac) to leaf versus wood production in forest ecosystems. Also shown (c) is the relationship between effects of eCO2 on canopy leaf production and canopy total foliage N content in forest experiments. Leaf Nac data in (b) are N content of green leaves minus N resorption from previous year’s foliage.

Figure 7. Effects of elevated CO2 (eCO2) on (a) annual aboveground net primary production (ANPP) , (b) corresponding nitrogen acquisition (Nac) and (c) N concentration ([N]) in relation to the number of years of experimental CO2 exposure (n = 8 for year 1 - 7; n = 7 for year 8 - 10; n = 5 for year 11). P values provided are based on t-tests of all individual temporal response trends (n = 8) while r2 values are based on the averaged data shown in the figure. Only experiments with data for ≥ 7 years were included (see Table 2).

Figure 8. Effects of elevated CO2 (eCO2) on (a) aboveground net primary production (ANPP) and (b) corresponding nitrogen acquisition (Nac) and (c) N concentration ([N]) in This article is protected by copyright. All rights reserved.

Accepted Article

relation to the number of years of experimental CO2 exposure at each FACE site. Annual N fertilization amounts are given in the figure legend. Solid and dashed lines have positive and negative slopes, respectively. LN, lack of (BioCON) or low N fertilization (Swiss-FACE); HN, presence of (BioCON) or high N fertilization (Swiss-FACE).









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