Why are nitrogen concentrations in plant tissues lower under elevated CO2?

A critical examination of the hypotheses.

Daniel R. Taub1, & Xianzhong Wang2

1Biology Department and Environmental Studies Program, Southwestern University,

1001 East University Avenue, Georgetown TX 78626, USA. 2Department of Biology,

Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA.

Author for correspondence- Daniel R. Taub (taubd@southwestern.edu),

Telephone: 1 512 863-1583 facsimile: 1 512 863-1696

Running title: Elevated CO2 and plant nitrogen concentrations

Abstract

Plants grown under elevated atmospheric [CO2] typically have decreased tissue

concentrations of N compared with plants grown under current ambient [CO2]. The

physiological mechanisms responsible for this phenomenon have not been definitely

established, although a considerable number of hypotheses have been advanced to

account for it. In this review we discuss and critically evaluate these hypotheses. One

contributing factor to the decreases in tissue N concentrations clearly is dilution of N by

increased photosynthetic assimilation of C. In addition, studies on intact plants show

strong evidence for a general decrease in the specific uptake rates (uptake per unit mass

or length of root) of N by roots under elevated CO2, This decreased root uptake appears

likely to be the result both of decreased N demand by shoots and of decreased ability of

the soil-root system to supply N. The best-supported mechanism for decreased N supply

is a decrease in transpiration-driven mass flow of N in soils due to decreased stomatal

conductance at elevated CO2, although some evidence suggests that altered root system

architecture may also play a role. There is also limited evidence suggesting that under

elevated CO2, plants may exhibit increased rates of N loss through volatilization and/or

root exudation, further contributing to lowering tissue N concentrations.

Key Words: carbon dioxide, dilution, elevated CO2, graphical vector analysis, nitrogen,

plants, root uptake, tissue concentrations

Growth of plants at atmospheric concentrations of carbon dioxide (CO2) greater

than the current ambient can greatly affect plant tissue chemistry (Loladze 2002, Poorter

et al. 1997). One of the most commonly seen effects is a decrease in the dry mass

concentration of N (Nm). Cotrufo et al. (1998), synthesizing data from a broad range of

studies, found mean decreases in Nm of 14% in aboveground tissues and 9% in roots.

This compares closely with the findings of other data syntheses, which have found

elevated [CO2] mediated decreases in Nm of 16.4% for leaves of woody plants (Curtis and

Wang 1998), 11% and 14% respectively for leaves of gymnosperms and angiosperms in

open-top chamber experiments (Norby et al. 1999), 12.9% for leaves in free-air carbon

dioxide enrichment (FACE) experiments (Ainsworth and Long 2005), 14% for seeds

(Jablonski et al. 2002) and 9-15% for the edible portions of several major food crops

(although only 1.4% for soybean; Taub et al. 2008). Such decreases in Nm can have

important implications not only for plant physiological processes, but for food chains, as

the performance of insect herbivores often decreases with decreases in Nm (Bezemer and

Jones 1998; Zvereva and Kozlov 2006), and changes in plant tissue quality under

elevated CO2 can affect herbivore population dynamics (Whittaker 1999).

While broad literature surveys have consistently found mean decreases in Nm of

approximately 10 - 15% for plants grown at elevated [CO2], there is considerable

variability in the results of individual studies. Yin (2002) found that, across a wide range

of studies, the elevated [CO2] effect on leaf Nm ranged from a 56% decrease to a 20%

increase, while Norby et al. (1999) found a range from a decrease of 35% to an increase

of 20% for the leaves of trees grown in open-top chambers. A number of factors have

been identified that partially explain this variation among studies. Yin (2002) found that

the effect of elevated [CO2] was greatest for woody deciduous species and that decreases

in Nm under elevated CO2 were most pronounced at high light levels, high temperatures

and large pot sizes. Yin (2002) and Taub et al. (2008) both found that the effect of [CO2]

on Nm was reduced by N fertilization. Several studies have reported that the effect of

elevated [CO2] on Nm is less for nitrogen-fixing species than for other types of plants

(Cotrufo et al. 1998; Jablonski et al. 2002; Taub et al. 2008). Ainsworth and Long (2005)

and Taub et al. (2008) found that the effect of elevated CO2 on Nm increased under ozone

stress (although see Taub et al., 2008 for divergent results for soybean).

While it has been well established that elevated [CO2] typically decreases Nm, the

mechanisms by which this occurs are not certain, although a large number of hypotheses

have been advanced to explain the phenomenon. While there have been a few attempts to

summarize and evaluate several of these hypotheses (Conroy and Hocking 1993; Gifford

et al. 2000; Pang et al. 2006), the current review aims to provide a critical and more

comprehensive evaluation of these hypotheses than has been available previously.

This review focuses on factors at the plant level that may affect Nm. Elevated

[CO2] and other global changes are likely to additionally affect Nm by affecting

ecosystem processes that influence N availability to plants, but this is beyond the scope

of this review; we refer readers to several recent reviews of these topics (Barnard et al.

2005; Luo et al. 2006; Reich et al. 2006).

Dilution Hypotheses

Perhaps the most frequently mentioned hypothesis about the decrease in Nm under

elevated [CO2] is that it results from dilution due to accumulation of non-structural

carbohydrates (NSC; Table 1, Hypothesis 1). Dilution by plant secondary compounds has

also been proposed as a possible mechanism for decreased Nm under elevated CO2 (Table

1, Hypothesis 2). Most authors who have mentioned dilution have not described the

phenomenon in detail; it is therefore uncertain precisely what process is envisioned. Here

we distinguish two different meanings of “dilution”, which we call biomass dilution and

functional dilution.

Biomass dilution occurs whenever the increase in total biomass of a plant or

organ under elevated CO2 relative to growth under ambient CO2 is greater than the

corresponding increase in total N. One tool that may be usefully applied to examining

biomass dilution is graphical vector analysis (GVA; (Haase and Rose 1995; Koricheva

1999). GVA is a technique that allows simultaneous comparison of concentration and

content of a chemical component (e.g., N or NSC) and the biomass of the plant organ that

contains the component (e.g., leaf) in an integrated graph, thus allowing detection of

dilution effects. Our methodology for constructing the vector diagram was modified from

Haase and Rose (1995). We extracted results from publications that contained data on

biomass and on leaf N and NSC contents. We superimposed the vector diagram for N

over the one for NSC to compare their changes in concentration and content relative to

changes in leaf mass under elevated CO2 (Figure 1).

The effect of elevated CO2 on N and NSC is determined by the direction and

magnitude of each vector (the arrows in Figure 1). A vector pointing downward and

toward the right indicates biomass dilution, while one pointing upward and to the right

indicates a concentration effect, in which a constituent increases to a greater extent under

elevated CO2 than does biomass as a whole. A horizontal vector pointing right indicates a

constituent that increases proportionally with increasing biomass. The length of a vector

indicates the magnitude of responsiveness to elevated CO2.

Figure 1 shows four distinct patterns of response to elevated CO2 in N and NSC.

Pattern 1, for Betula pendula, shows a response in which N is diluted and NSC are

concentrated under elevated CO2. Pattern 2, for Pinus plaustris at high soil water

potential, shows a response in which NSC are concentrated under elevated CO2, while N

decreases not only in concentration but in absolute content as well. This suggests that a

mechanism in addition to biomass dilution is contributing to decreasing Nm. Pattern 3, for

Pinus palustris at low soil water potential, shows dilution of N, but there is very little

increase in the concentration of NSC, suggesting that other biomass constituents are

largely responsible for the dilution of N. Pattern 4, for Xanthium strumarium, shows

dilution of both N and NSC by increased biomass.

These results demonstrate that one important reason of lower N under elevated

CO2 is the relatively small increase of its content compared to other components, i.e.

biomass dilution. These results also demonstrate that NSC is in some cases an important

component of the diluting biomass. In no case, however, does NSC increase sufficiently

to entirely account for the dilution of N. This finding seems likely to hold in general.

Poorter et al. (1997) analyzed the chemical composition of the leaves of 27 species grown

under elevated CO2 across a wide range of experimental protocols. They found that Nm

was decreased under elevated CO2 by an average of 17% on a total biomass basis and by

10% under a NSC-free biomass basis. The content of protein (the major nitrogenous

component of leaves) declined not only relative to NSC, but relative to all other classes of

organic compounds, including structural carbohydrates, soluble phenolics, lipids, organic

acids and lignin. Biomass dilution of N under elevated CO2 may therefore be near-

ubiquitous, but NSC are responsible for only a portion of this effect.

Additional insight into the mechanism of dilution can be obtained through the

concept of functional dilution of N. This concept is based on the functional balance

concept which envisions tissue N concentrations as dependent on the relative activities of

shoots and roots:

ss

rrm f

fN

σσ

∝

where fr and fs are the fractions of plant mass in roots and shoots, respectively, and σr and

σs are the mass-based specific activities of roots and shoots in acquiring N and

photosynthate, respectively (c.f BassiriRad et al. 2001, Davidson 1969, Hilbert 1990).

Within this model, there are three possible causes of a decrease in Nm: a shift in

allocation of biomass toward shoots, a decrease in specific root activity, or an increase in

shoot specific activity. A decrease in Nm due to increased shoot specific activity can be

regarded as functional dilution; Nm declines because of the accumulation of additional

photosynthate by shoots.

If we can accept photosynthetic rate as the equivalent of shoot specific activity,

functional dilution of N under elevated CO2 appears near-ubiquitous, as enhancement of

photosynthetic rates for plants grown under elevated CO2 is consistently observed on

either a leaf area or leaf mass basis (Ainsworth and Long 2005, Curtis and Wang 1998,

Ellsworth et al. 2004, Norby et al. 1999). The question arises of whether, within the

functional balance concept, dilution is solely responsible for decreased Nm under elevated

CO2, or whether altered allocation and/or decreased specific root activity play a role as

well.

Altered allocation does not appear likely to contribute to decreased Nm; to the

extent that elevated CO2 affects allocation it appears generally to increase root mass

relative to shoot mass (Luo et al. 2006, Norby 1994, Poorter and Nagel 2000). This

should tend to increase, rather than decrease, Nm.

Decreased specific root activity appears more likely than altered allocation to play

a role in decreasing Nm. While a number of experiments have measured the effects of

elevated CO2 on root uptake kinetics in solution (see references in BassiriRad et al.

2001), data on specific root uptake of N (i.e. uptake per unit root mass or length) under

elevated CO2 for intact plants rooted in solid media (i.e. not in hydroponics) are fairly

limited. Table 2 shows the results of all such experiments that we have identified. The

consistent trend toward decreased specific uptake is quite remarkable (although the trends

were often not statistically significant within individual experiments), as is the overall

mean decrease in uptake rate of 16.4%. This result is in striking contrast to the effects of

elevated CO2 on uptake of N from solution. Reviews of the literature on short-term

(minutes to hours) uptake of NH4+ and NO3

- from solution have concluded that results

from individual studies are highly variable, with uptake variously increased, decreased or

unchanged under elevated CO2 (BassiriRad et al. 2001, Luo et al. 1998). Studies that

have measured longer-term uptake of N (days to weeks) in plant grown in hydroponics

have also shown variable results, with increases in specific uptake of N under elevated

CO2 appearing more common than decreases (Chu et al. 1992, Gloser et al. 2002, Roumet

et al. 1996). It therefore appears that the factors that lead to decreased specific N uptake

from soils under elevated CO2 are ones not present in hydroponics. This might include

aspects of root system architecture and mycorrhizal status or factors affecting the

movement of N through soils to plant roots (see the following section for an extended

discussion). This suggestion is also consistent with the finding that decreases in Nm under

elevated CO2 are larger in soils than in hydroponics (Poorter et al. 1997). In any case,

decreased rates of root specific uptake of N appear likely to play a role in the decreased

Nm of plants grown in soil.

Another line of evidence that suggests that dilution is not solely responsible for

decreasing Nm under elevated CO2 is the pattern of response of mineral elements other

than N. With either functional dilution or biomass dilution, all elements other than the

elements assimilated through photosynthesis (C, H and O) should be diluted equally

(Loladze 2002). Figure 2 shows that significant decreases are seen in elevated CO2 for

four of five macronutrient elements, but that there are significant differences among the

elements in the effect of CO2. In particular, N is affected by CO2 more than P and K are.

This strongly suggests that the effects of elevated CO2 on mineral element concentrations

are mediated through factors involved in uptake and/or metabolism of these elements, not

simply through dilution.

Hypotheses of decreased nitrogen uptake (source effects)

A variety of hypotheses have been advanced that propose mechanisms by which

growth at elevated [CO2] might decrease acquisition of N. These mechanisms can be

divided into those that affect N uptake by affecting the ability of the soil-root system to

supply N (source effects), and those that affect demand for N by the plant, with

subsequent effects on N uptake (demand effects). In this section we discuss source-driven

effects on N acquisition, with demand-driven mechanisms reserved for the following

section.

One of the most consistent effects of growth at elevated CO2 is a decrease in

stomatal conductance (Ainsworth and Rogers 2007, Wullschleger et al. 2002), leading to

decreased transpiration. Several authors have suggested that this may decrease uptake of

those nutrients for which mass flow through soil plays a major role in uptake, including N

(as nitrate), Ca, Mg and S (Table 1; Line 3; Barber 1984, Marschner 1995).

In support of this hypothesis, several studies have found elevated CO2 to have

similar effects on both transpiration and plant N. Del Pozo et al. (2007), manipulating

both N supply and [CO2] in wheat, found transpiration and leaf N (both on a leaf area

basis) to be positively correlated. Polley et al. (1999) found that elevated CO2 decreased

both whole-plant transpiration and whole-plant N accretion in two C3 perennials.

In either of these studies it is not clear whether transpiration has led to decreased

plant N, or whether decreased photosynthetic capacity (manifested by decreased leaf N)

has led to decreased stomatal conductance and transpiration (Drake et al. 1997). This

caveat does not hold for a study by McDonald et al. (2002), who measured the effect of

elevated CO2 on N uptake rates. They found that over a seven day period, transpiration

per gram of root in Populus deltoides was both decreased by 20.4% under elevated CO2

and positively correlated with N uptake per gram of root.

Additional support for a relationship between elevated CO2-mediated decreases in

transpiration and in Nm can be found by comparing the responses to elevated CO2 of

various soil-derived elements. Figure 2 shows that decreases in concentration under

elevated CO2 are largest for those macronutrients that are supplied to roots by

transpiration-driven mass flow (N, Mg and Ca) and least for those most dependent on

diffusion through soil (P and K). This suggests two possible interpretations. Biomass

dilution may decrease the concentrations of all soil-derived elements, while mobile

elements are additionally decreased due to restricted mass flow. Alternatively, dilution

may decrease the concentration of all soil-derived elements, but this may be partially

ameliorated for diffusion-limited elements by increased soil water content allowing more

rapid diffusion (Van Vuuren et al. 1997).

Several authors have suggested that growth at elevated CO2 may lead to root

system architectures that are less efficient at taking up nutrients, including N (Table 1;

Hypothesis 4). Pritchard and Rogers (2000) presented evidence that under elevated CO2,

root systems often exhibit increased growth of lateral vs. primary roots, leading to

shallower rooting, and suggested that this would lead to decreased efficiency of nutrient

uptake. Berntson (1994), modelling root systems of Senecio vulgaris, found that changes

in root system architecture under elevated CO2 led to decreased efficiency of soil

exploitation. Whether this finding would hold for other species and under different

growth conditions is unknown.

Another mechanism that might potentially affect N uptake rates is altered uptake

capacity of individual roots (Table 1; hypothesis 5). The expectation of most authors has

been that elevated CO2 would increase rather than decrease uptake capacity, by providing

additional photosynthate to roots for the energy demanding processes of N uptake

(BassiriRad et al. 1996, 2001). Studies of root uptake of NH4+ and NO3

- from solution

have, however, yielded highly variable results (Luo et al. 1998), with a majority of the

experimental observations summarized by BassiriRad et al. (2001) showing decreases in

NH4 and NO3 uptake rates under elevated CO2. The mechanisms by which elevated CO2

might lead to such decreases are not known. The large variability seen for the effects of

elevated CO2 on root uptake capacity suggest that this factor is not primarily responsible

for the decrease in Nm typically seen under elevated CO2, but may contribute to the

variation seen in magnitude of the Nm response.

Altered mycorrhizal status under elevated CO2 has also frequently been

mentioned as a mechanism that might affect plant nutrient status (Table 1; Hypothesis 6).

Growth at elevated CO2 often increases the proportion of roots colonized by mycorrhizal

fungi (Treseder 2004). It may also have greater positive effects on the mass of

mycorrhizal fungi than of their associated plants (BassiriRad et al. 2001; though see

Staddon et al. 2002), and increase the growth of extraradical mycelium more than it

increases the growth of fungal hyphae in direct association with plant roots (Alberton et

al. 2005). These changes collectively could potentially increase nutrient uptake efficiency

under elevated CO2 by the fungal-plant system, as fungal hyphae have greater uptake

capacity on a mass basis than plant roots (BassiriRad et al. 2001). Alternatively, the

greater fungal mass under elevated CO2 might provide a larger sink for minerals, so that

less is translocated to plants (Alberton et al. 2005, BassiriRad et al. 2001). BassiriRad et

al. (2001) found that, under elevated CO2, mycorrhizal plants typically had higher Nm

than non-mycorrhizal plants. This suggests that effects on mycorrhizae are unlikely to be

responsible for the decrease in Nm typically found under elevated CO2. Differences in the

extent of mycorrhizal development may, however, explain some of the variability found

in the effect of elevated CO2 on Nm, with colonization by mycorrhizal fungi partially

ameliorating the decreases in Nm that are typically seen.

Hypotheses of decreased nitrogen demand

Plants can sustain growth at a lower Nm under elevated than under ambient CO2,

as reflected in a lower critical foliar N concentration (the concentration at which biomass

production is 90% of maximum; Conroy 1992) and increased plant nitrogen use

efficiency (Stitt and Krapp 1999). Assuming that N uptake is at least partially regulated

by demand (BassiriRad et al. 2001), this decreased N requirement can lead to decreased

whole-plant Nm (Table 1, Hypothesis 7).

Increased plant N efficiency for biomass production under elevated CO2 is almost

certainly the product of an increased photosynthetic N use efficiency (PNUE). This

increased PNUE appears to be a result both of the effect of [CO2] on the efficiency of

carboxylation of RUBP and of decreased investment in photosynthetic and

photorespiratory enzymes (Davey et al. 1999, Gifford et al. 2000, Stitt and Krapp 1999).

While there have been suggestions that decreased accumulation of photosynthetic

enzymes under elevated CO2 result from a general decline in leaf N status (Stitt and

Krapp 1999), there is also evidence for a specific downregulation of photosynthetic

enzymes mediated by a sugar-sensing mechanism (Moore et al. 1999).

Photosynthetic enzymes, particularly RUBISCO, make up a large fraction of total

leaf N in C3 species (Evans and Seemann 1989). A number of authors have therefore

suggested that downregulation of photosynthesis may be partially or largely responsible

for the effects of elevated CO2 on Nm in leaves and other photosynthetic tissues (Table 1,

Hypothesis 8). Fangmeier et al. (1999, 2000, 2002) have pointed out that downregulation

of photosynthetic enzymes in leaves may also decrease N availability to organs such as

seeds and tubers that obtain N translocated from catabolised proteins in leaves. Increased

PNUE and decreased demand for photosynthesis may therefore explain, at least in part,

decreased Nm in a variety of plant tissues.

Elevated CO2-mediated nitrogen loss hypothesis

While many authors have considered possible CO2 effects on N acquisition, only

one study that we are aware of has considered the possibility that elevated CO2 decreases

Nm by affecting the rate of plant loss of N (Table 1; Hypothesis 9). Pang et al. (2006)

examined N relations in rice grown in pots of nutrient solution under free-air carbon

dioxide enrichment. They documented that N loss per pot was greater under elevated than

ambient CO2 at both high and low N supply. They attributed this N loss to volatilization

of NH3 from senescing plant tissues, and possibly to root exudation of organic N, and

suggested that these increases in N loss were responsible for the observed decreases in

Nm.

Closer examination of their data suggests that other mechanisms must have also

been operating. In their experiment, whole-plant Nm (recalculated from their data) was

decreased under elevated CO2 by 26.4% and 26.5% at low and high N, respectively. Had

all of the N lost from pots been retained in the plant tissues, Nm would still have been

decreased under elevated CO2 by 21.4 and 19.1% at low N and high N, respectively.

Effects of elevated CO2 on N loss therefore may have played a role in the observed

decrease in Nm under elevated CO2, but other mechanisms were probably more

important. Whether the effect of elevated CO2 on N loss is particular to this system also

appears to be unknown. Loss of N from senescing leaves does not appear likely to

explain the decreases in Nm seem in many experiments in young, non-senescent tissues.

Hypothesis of ontogenetic drift in N concentration

Coleman et al. (1993) proposed that the observed effects of elevated CO2 on Nm

may be ontogenetic rather than functional. Plants grow more quickly under elevated than

ambient CO2. Comparisons between elevated and ambient CO2-grown plants of the same

age are therefore comparisons between plants of different sizes. For seedlings, Nm often

decreases during early ontogeny. Differences in Nm between elevated and ambient-CO2

grown plants may therefore simply reflect the fact that the plants are of different sizes

rather than any specific effect of CO2 on plant physiology.

Coleman et al. (1993) presented data showing that for two annual species,

seedlings grown under elevated CO2 had lower Nm than ambient-grown seedlings when

compared at identical ages. When the comparison was made between elevated and

ambient-grown plants at the same size, the difference disappeared.

Subsequent studies have in general not fully replicated this finding. The effect of

elevated CO2 in some studies is less when compared at the same size rather than the same

age, but allometric analyses typically show differences in Nm between elevated and

ambient grown plants that are independent of plant size (Bernacchi et al. 2007, Harmens

et al. 2001, Lutze and Gifford 1998, Marriott et al. 2001). Some studies also show an

effect of CO2 on Nm, with little or no evidence for ontogenetic drift in Nm (den Hertog et

al. 1996, Lutze and Gifford 1998, Marriott et al. 2001).

Ontogenetic drift also appears unlikely to explain elevated CO2-mediated

differences in Nm seen in perennial plants after more than one season of growth (e.g.

Curtis and Wang 1998, Ellsworth et al. 2004) or between seeds compared at full maturity

(e.g. Jablonski et al. 2002, Taub et al. 2008). Overall, ontogenetic drift in Nm appears

likely to play only a minor role in creating the general pattern of decreased Nm under

elevated CO2.

Conclusions

A variety of physiological effects of elevated CO2 on plants have been proposed

that could potentially influence plant tissue concentrations of N. It is possible that many

or even all of these mechanisms genuinely operate in plants, at least for particular species

or under particular environmental conditions. Some mechanisms, such as increases in

mycorrhizae or increased capacity for N uptake at root surfaces, appear likely to affect

Nm in a positive direction. Other mechanisms, such as biomass dilution, decreased

transpiration, decreased efficiency of root architecture and increased N loss are likely to

lead to decreased Nm.

A substantial number of literature surveys are in agreement that the mean effect of

elevated CO2 on Nm is a 10-15% decrease (Ainsworth and Long 2005, Cotrufo et al.

1998, Curtis and Wang 1998, Jablonski et al. 2002, Norby et al. 1999, Taub et al. 2008).

This suggests that physiological changes leading to decreased Nm under elevated CO2

predominate in their effects over factors that would tend to increase Nm.

We suggest that the predominant mechanisms by which elevated CO2 affects Nm

are dilution of N in plant tissues by increased concentrations of compounds derived from

photosynthate, and decreases in root specific N uptake. Decreased specific uptake is

likely due both to decreased demand by the plant (due to increased photosynthetic

nitrogen use efficiency and to downregulation of photosynthetic enzymes) and to

decreased ability of the soil-root system to supply N. While a number of mechanisms

have been proposed that might account for this decreased N supply, we suggest that

decreased transpiration-driven mass flow of N is the mechanism that is best supported by

the available evidence.

Two additional mechanisms appear particularly worthy of additional

investigation. Both decreased efficiency of root system architecture (Berntson 1994) and

increased loss of N by plants under elevated CO2 (Pang et al. 2006) have received

experimental support as possible mechanisms contributing to decreased Nm. However, in

each case this is based (to the best of our knowledge) on a single study, and additional

studies are clearly needed if these mechanisms are to be substantiated.

Acknowledgments

We thank the Cullen Fund of Southwestern University for financial support (to DRT).

Lisa Anderson helped in obtaining literature. Jim Coleman and Kelly McConnaughay

provided helpful discussion of their research, and the reviewers and editor provided

helpful suggestions on the manuscript.

References Adams DC, Gurevitch J, Rosenberg MS (1997). Resampling tests for meta-analysis of ecological data. Ecology 78, 1277-1283. Ainsworth EA, Long SP (2005). What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2. New Phytologist 165, 351-372. Ainsworth EA, Rogers A (2007). The response of photosynthesis and stomatal conductance to rising [CO2]: mechanisms and environmental interactions. Plant, Cell and Environment 30, 258-270. Alberton O, Kuyper TW, Gorissen A (2005). Taking mycocentrism seriously: mycorrhizal fungal and plant responses to elevated CO2. New Phytologist 167, 859-868. Barber SA (1984). Soil Nutrient Bioavailability: A Mechanistic Approach. Wiley, New York. Barnard R, Leadley PW, Hungate BA (2005) Global change, nitrification, and denitrification: A review. In: Global Biogeochemical Cycles, pp. GB1007. BassiriRad H, Griffin KL, Strain BR, Reynolds JF (1996). Effects of CO2 enrichment on growth and root 15NH4

+ uptake rate of loblolly pine and ponderosa pine seedlings. Tree Physiology 16, 957-962. BassiriRad H, Gutschick VP, Lussenhop J (2001). Root system adjustments: regulation of plant nutrient uptake and growth responses to elevated CO2. Oecologia 126, 305-320. Bernacchi CJ, Thompson JN, Coleman JS, McConnaughay KDM (2007). Allometric analysis reveals relatively little variation in nitrogen versus biomass accrual in four plant species exposed to varying light, nutrients, water and CO2. Plant, Cell and Environment 30, 1216-1222. Berntson GM (1994). Modelling root architecture: Are there tradeoffs between efficiency and potential of resource acquisition? New Phytologist 127, 483-493. Berntson GM, Bazzaz FA (1996). Belowground positive and negative feedbacks on CO2 growth enhancement. Plant and Soil 187, 119-131. Bezemer TM, Jones TH (1998). Plant-insect herbivore interactions in elevated atmospheric CO2: quantitative analyses and guild effects. Oikos 82, 212-222. Chu CC, Coleman JS, Mooney HA (1992). Controls of biomass partitioning between roots and shoots: Atmospheric CO2 enrichment and the acquisition and allocation of carbon and nitrogen in wild radish. Oecologia 89, 580-587. Coleman JS, Bazzaz FA (1992). Interacting effects of elevated CO2 and temperature on growth and resource use of co-occurring annual plants. Ecology 73, 1244-1259. Coleman JS, McConnaughay KDM, Bazzaz FA (1993). Elevated CO2 and plant nitrogen-use: is reduced tissue nitrogen concentration size-dependent. Oecologia 93, 195-200. Conroy J, Hocking P (1993). Nitrogen nutrition of C3 plants at elevated atmospheric CO2 concentrations. Physiologia Plantarum 89, 570-576. Conroy JP (1992). Influence of elevated atmospheric CO2 concentrations on plant nutrition. Australian Journal of Botany 40, 445-456. Cotrufo MF, Ineson P, Scott A (1998). Elevated CO2 reduces the nitrogen concentration of plant tissues. Global Change Biology 4, 43-54.

Curtis PS, Wang X (1998). A meta-analysis of elevated CO2 effects on woody plant mass, form and physiology. Oecologia 113, 299-313. Davey PA, Parson AJ, Atkinson L, Wadge K, Long SP (1999). Does photosynthetic acclimation to elevated CO2 increase photosynthetic nitrogen-use efficiency? A study of three native UK grassland species in open-top chambers. Functional Ecology 13 (Suppl 1.), 21-28. Davidson RL (1969). Effect of root/leaf temperature differentials on root/shoot ratios in some pasture grasses and clover. Annals of Botany 33, 561-569. Del Pozo A, Pérez P, Gutiérrez D, Alonso A, Morcuende R, Martínez-Carrasco R (2007). Gas exchange acclimation to elevated CO2 in upper-sunlit and lower-shaded canopy leaves in relation to nitrogen acquisition and partitioning in wheat grown in field chambers. Environmental and Experimental Botany 59, 371-380. den Hertog J, Stulen I, Fonseca F, Delea P (1996). Modulation of carbon and nitrogen allocation in Urtica dioca and Plantago major by elevated CO2: Impact of accumulation of nonstructural carbohydrates and ontogenetic drift. Physiologia Plantarum 98, 77-88. Drake BG, Gonzàlez-Meler, Long SP (1997). More efficient plants: A consequence of rising atmospheric CO2? Annual Review of Plant Physiology and Molecular Biology 48, 609-639. Ellsworth DS, Reich PB, Namburg ES, Koch GW, Kubiske ME, Smith SD (2004). Photosynthesis, carboxylation and leaf nitrogen responses of 16 species to elevated pCO2 across four free-air CO2 enrichment experiments in forest, grassland and desert. Global Change Biology 10, 2121-2138. Evans JR, Seemann JR (1989). The allocation of protein nitrogen in the photosynthetic apparatus: Costs, consequences, and control. In: Briggs WR, ed. Photosynthesis. Alan R. Liss, New York. pp. 183-205. Fangmeier A, Chrost B, Högy P, Krupinska K (2000). CO2 enrichment enhances flag leaf senescence in barley due to greater grain nitrogen sink capacity. Environmental and Experimental Botany 44, 151-164. Fangmeier A, De Temmerman L, Black C, Persson K, Vorne V (2002). Effects of elevated CO2 and/or ozone on nutrient concentrations and nutrient uptake of potatoes. European Journal of Agronomy 17, 353-368. Fangmeier A, De Temmerman L, Mortensen L, Kemp K, Burke J, Mitchell R et al. (1999). Effects of nutrients on grain quality in spring wheat crops grown under elevated CO2 concentrations and stress conditions in the European multiple-site experiment 'ESPACE-wheat'. European Journal of Agronomy 10, 215-229. Gifford RM, Barrett DJ, Lutze JL (2000). The effects of elevated [CO2] on the C:N and C:P mass ratios of plant tissues. Plant and Soil 224, 1-14. Gloser V, Frehner M, Lüscher A, Nösberger J, Hartwig UA (2002). Does the response of perennial ryegrass to elevated CO2 concentration depend on the from of the supplied nitrogen? Biologia Plantarum 45, 51-58. Haase DL, Rose R (1995). Vector analysis and its use for interpreting plant nutrient shifts in response to silvicultural treatments. Forest Science 41, 54-66. Harmens H, Marshall C, Stirling CM, Farrar JF (2001). Partitioning and efficiency of use of N in Dactylis glomerata as affected by elevated CO2: interaction with N supply. International Journal of Plant Sciences 162, 1267-1274.

Hilbert DW (1990). Optimization of plant root:shoot ratios and internal nitrogen concentration. Annals of Botany 66, 91-99. Israel DW, Rufty Jr. TW, Cure JD (1990). Nitrogen and phosphorus nutritional interactions in a CO2 enriched environment. Journal of Plant Nutrition 13, 1419-1433. Jablonski LM, Wang X, Curtis PS (2002). Plant reproduction under elevated CO2 conditions: a meta-analysis of reports on 79 crop and wild species. New Phytologist 156, 9-26. Koricheva J (1999). Interpreting phenotypic variation in plant allelochemistry: problems with the use of concentrations. Oecologia 199, 467-473. Kuehny JS, Peet MM, Nelson PV, Willits DH (1991). Nutrient dilution by starch in CO2-enriched Chysanthemum. Journal of Experimental Botany 42, 711-716. Larigauderie A, Reynolds JF, Strain BR (1994). Root response to CO2 enrichment and nitrogen supply in loblolly pine. Plant and Soil 165, 21-32. Lewis JD, Wang XZ, Griffin KL, Tissue DT (2002). Effects of age and ontogeny on photosynthetic responses of a determinate annual plant to elevated CO2 concentrations. Plant, Cell and Environment 25, 359-368. Loladze I (2002). Rising atmospheric CO2 and human nutrition: toward globally imbalanced plant stoichiometry? Trends in Ecology and Evolution 17, 457-461. Luo Y, Hui D, Zhang D (2006). Elevated CO2 stimulates net accumulations of carbon and nitrogen in land ecosystems: A meta-analysis. Ecology 87, 53-63. Luo Y, Reynolds J, Wang Y, Wolfe D (1998). A search for predictive understanding of plant responses to elevated [CO2]. Global Change Biology 5, 143-156. Lutze JL, Gifford RM (1998). Aquistion and allocation of carbon and nitrogen by Danthonia richardsonii in response to restricted nitrogen supply and CO2 enrichment. Plant, Cell and Environment 21, 1133-1141. Lynch JP, St.Clair SB (2004). Mineral stress: the missing link in understanding how global climate change will affect plants in real world soils. Field Crops Research 90, 101-115. Marriott DJ, Stirling CM, Farrar J (2001). Constraints to growth of annual nettle (Urtica urens) in an elevated CO2 atmosphere: Decreased leaf area ratio and tissue N cannot be explained by ontogenetic drift of mineral N supply. Physiologia Plantarum 111, 23-32. Marschner H (1995). Mineral Nutrition of Higher Plants. (2nd ed.). Academic Press, London. McDonald EP, Erickson JE, Kruger EL (2002). Can decreased transpiration limit plant nitrogen acquisition in elevated CO2? Functional Plant Biology 29, 1115-1120. Moore BD, Cheng S-H, Sims D, Seemann JR (1999). The biochemical and molecular basis for photosynthetic acclimation to elevated atmospheric CO2. Plant, Cell and Environment 22, 567-582. Norby RJ (1994). Issues and perspectives for investigating root responses to elevated atmospheric carbon dioxide. Plant and Soil 165, 9-20. Norby RJ, Wullschleger SD, Gunderson CA, Johnson DW, Ceulemans R (1999). Tree responses to rising CO2 in field experiments: implications for the future forest. Plant, Cell and Environment 22, 683-714.

Pang J, Zhu J-G, Xie ZB, Liu G, Zhang Y-L, Chen G-P et al. (2006). A new explanation of the N concentration decrease in tissues of rice (Oryza sativa L.) exposed to elevated atmospheric pCO2. Environmental and Experimental Botany 57, 98-105. Pettersson R, McDonald AJS (1992). Effects of elevated carbon dioxide concentration on photosynthesis and growth of small birch plants (Betula pendula Roth.) at optimal nutrition. Plant, Cell and Environment 15, 911-919. Polley HW, Johnson HB, Tischler CR, Torbert HA (1999). Links between transpiration and plant nitrogen: Variation with atmospheric CO2 concentration and nitrogen availability. International Journal of Plant Sciences 160, 535-542. Poorter H, Nagel O (2000). The role of biomass allocation in the growth response of plants to different levels of light, CO2, nutrients and water: a quantitative review. Australian Journal of Plant Physiology 27, 595-607. Poorter H, van Berkel Y, Baxter R, den Hertog J, Dijkstra P, Gifford RM et al. (1997). The effect of elevated CO2 on the chemical composition and construction costs of leaves of 27 C3 species. Plant, Cell and Environment 20, 472-482. Pritchard SG, Rogers HH (2000). Spatial and temporal deployment of crop roots in CO2-enriched environments. New Phytologist 147, 55-71. Reich PB, Hungate BA, Luo Y (2006). Carbon-nitrogen interactions in terrestrial ecosystems in response to rising atmospheric carbon dioxide. Annual Review of Ecology, Evolution and Systematics 37, 611-636. Rogers HH, Peterson CM, McCrimmon JN, Cure JD (1992). Response of plant roots to elevated atmospheric carbon dioxide. Plant, Cell and Environment 15, 749-752. Roumet C, Bel MP, Sonie L, Jardon F, Roy J (1996). Growth response of grasses to elevated CO2: a physiological plurispecific analysis. New Phytologist 133, 595-603. Runion GB, Entry JA, Prior SA, Mitchell RJ, Rogers HH (1999). Tissue chemistry and carbon allocation in seedlings of Pinus palustris subjected to elevated atmospheric CO2 and water stress. Tree Physiology 19, 329-335. Staddon PL, Heinemeyer A, Fitter AH (2002). Mycorrhizas and global environmental change: research at different scales. Plant and Soil 244, 253-261. 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. Taub D, Miller B, Allen H (2008). Effects of elevated CO2 on the protein concentration of food crops: a meta-analysis. Global Change Biology 14, 565-575. Treseder KK (2004). A meta-analysis of mycorrhizal responses to nitrogen, phosphorus, and atmospheric CO2 in field studies. New Phytologist 164, 347-355. Van Vuuren MMI, Robinson D, Fitter AH, Chasalow SD, Williamson L, Raven JA (1997). Effects of elevated atmospheric CO2 and soil water availability on root biomass, root length and N, P and K uptake by wheat. New Phytologist 135, 455-465. Vivin P, Martin F, Guehl J-M (1996). Acquisition and within-plant allocation of 13C and 15N in CO2-enriched Quercus robur plants. Physiologia Plantarum 98, 89-96. Whittaker JB (1999). Impacts and responses at population level of herbivorous insects to elevated CO2. European Journal of Entomology 96, 149-156. Wong S-C (1990). Elevated atmospheric partial pressure of CO2 and plant growth. II. Non-structural carbohydrate content in cotton plants and its effect on growth parameters. Photosynthesis Research 23, 171-180.

Wullschleger SD, Tschaplinski TJ, Norby RJ (2002). Plant water relations at elevated CO2- implications for water-limited environments. Plant, Cell and Environment 25, 319-331. Yin X (2002). Responses of leaf nitrogen concentration and specific leaf area to atmospheric CO2 enrichment: a retrospective synthesis across 62 species. Global Change Biology 8, 631-642. Zerihun A, Gutschick VP, BassiriRad H (2000). Compensatory roles of nitrogen uptake and photosynthetic N-use efficiency in determining plant growth response to elevated CO2: Evaluation using a functional balance model. Annals of Botany 86, 723-730. Zvereva EL, Kozlov MV (2006). Consequences of simultaneous elevation of carbon dioxide and temperature for plant-herbivore interactions: a metaanalysis. Global Change Biology 12, 27-41.

Table 1: Hypotheses to account for the decrease in tissue N concentrations under elevated

[CO2]. Papers cited discuss, but do not necessarily endorse, the hypotheses under

question.

Hypothesis Selected references

1 Dilution by carbohydrates 1, 2, 3

2 Dilution by secondary compounds 1

3 Decreased N uptake due to decreased transpiration 4, 5, 6, 7

4 Less efficient root architecture 8, 9, 10

5 Altered root uptake capacity 9, 11

6 Altered mycorrhizal status 11, 12

7 Increased N efficiency 13, 14

8 Downregulation of photosynthesis 1, 15, 16

9 Increased N loss 17

10 Ontogenetic drift of N concentration 18, 19

1(Gifford et al. 2000) 2(Kuehny et al. 1991) 3(Wong 1990) 4(Van Vuuren et al. 1997) 5(Del Pozo et al. 2007)

6(McDonald et al. 2002) 7(Conroy and Hocking 1993) 8(Pritchard and Rogers 2000)

9(Berntson and Bazzaz 1996) 10(Lynch and St.Clair 2004) 11(BassiriRad et al. 2001)

12(Alberton et al. 2005) 13(Stitt and Krapp 1999) 14(Conroy 1992) 15(Fangmeier et al. 1999)

16(Fangmeier et al. 2000) 17(Pang et al. 2006) 18(Coleman et al. 1993) 19(Bernacchi et al. 2007)

Paper Species Conditions

Method and duration of N-uptake measurements

Root specific uptake rate under elevated CO2 as % of that under ambient CO2

Coleman and Bazzaz (1992) Abutilon theophrasti 28 degrees Sequential harvests (20 days) 59.6% Coleman and Bazzaz (1992) Abutilon theophrasti 38 degrees Sequential harvests (20 days) 60.6% Coleman and Bazzaz (1992) Amaranthus retroflexus 28 degrees Sequential harvests (20 days) 56.6% Coleman and Bazzaz (1992) Amaranthus retroflexus 38 degrees Sequential harvests (20 days) 76.7%

Israel et al. (1990) Glycine max (non-nodulated) High N, High P

Single harvest (27 days) 89.2%

Israel et al. (1990) Glycine max (non-nodulated) High N, low P

Single harvest (27 days) 103.1%

Israel et al. (1990) Glycine max (non-nodulated) Low N, High P

Single harvest (27 days) 87.2%

Rogers et al. (1992) * Glycine max (non-nodulated)

Single harvest (18 days) 90.7%

Zerihun et al. (2000) Helianthus annuus Low N Sequential harvests (20 days) 71.4% Zerihun et al. (2000) Helianthus annuus Medium N Sequential harvests (20 days) 100.0% Zerihun et al. (2000) Helianthus annuus High N Sequential harvests (20 days) 76.9% Larigauderie et al. (1994) Pinus taeda Low N Sequential harvests (117 days) 79.2% Larigauderie et al. (1994) Pinus taeda High N Sequential harvests (117 days) 122.9% BassiriRad et al. (1996) Pinus taeda Uptake of 15NH4

+(48 hours) 82.6% BassiriRad et al. (1996) Pinus ponderosa Uptake of 15NH4

+(48 hours) 79.3% McDonald et al. (2002) Populus deltoides Uptake of 15NO3

- (7 days) 86.4% Vivin et al. (1996) Quercus robur Uptake of 15NO3

- (12 hours) 100.0% mean (across all studies) 83.6% median (across all studies) 82.6%

Table 2: Effect of growth under elevated CO2 on root specific uptake rates (i.e. uptake per gram root) in studies performed on intact plants rooted in solid media. When a time-series was reported, measurements are those from the period of greatest uptake rate. Sequential harvest techniques estimate uptake by integrating root mass (or area) and plant N content over the interval between two harvests at which these are measured. The single harvest technique uses a single harvest to obtain the root mass (or area) and N measurements at the end of the experiment. As Israel et al. (1990) point out, this is better thought of as an index of relative N uptake rather than a true estimate of specific uptake. * uptake per root surface area rather than weight.

Figure Legends

Figure 1. Graphical vector analysis of the effect of elevated CO2 on leaf concentrations of

N and non-structural carbohydrates (NSC) in four experiments. All data values are

percents relative to the value at ambient CO2, with the large diamond at (100,100)

representing the value of all measurements under ambient CO2. All other data points are

for plants under elevated CO2. Diagonal lines indicate relative leaf mass (i.e. leaf mass

under elevated CO2 as a percentage of that under ambient CO2). See text for further

explanation. Data is taken from: 1- Pettersson and McDonald (1992) 2,3- Runion et al.

(1999), -0.5 Mpa, -1.5Mpa water potential, respectively 4- Lewis et al. (2002).

Figure 2. Effect of elevated CO2 on macronutrient concentrations in the edible portions of

food crops of studies that measured all elements in the same tissues (n=42). Results are

from an unpublished meta-analysis of the relevant research literature (D. Taub and X.

Wang). Geometric means and bootstrapped 95% confidence limits (Adams et al. 1997).

Vertical dashed line indicates no effect of elevated CO2.

Figure 1:

Relative N content (open symbols), Relative NSC content (closed symbols)

60 80 100 120 140 160 180 200

Rel

ativ

e N

% (

open

sym

bols

), R

elat

ive

NSC

% (

clos

ed s

ymbo

ls)

60

80

100

120

140

160

180

200

1 - Betula pendula2 - Pinus palustris (-0.5 Mpa)3 - Pinus palustris (-1.5 Mpa)4 - Xanthium strumarium1 - Betula pendula2 - Pinus palustris (-0.5 Mpa)3 - Pinus palustris (-1.5 Mpa)4 - Xanthium strumarium

40 60 80 100

120

160

140

Relative Leaf Mass

180

Figure 2:

Percent decrease in concentration under elevated CO2

-25 -20 -15 -10 -5 0 5

Mg

Ca

K

P

N