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An overlooked carbon source for grassland soils: loss of structural carbon from stubble in response to elevated pCO2 and nitrogen supply Blackwell Publishing Ltd

Manuel K. Schneider1,2, Andreas Lüscher1,3, Emmanuel Frossard1 and Josef Nösberger1 1

Institute of Plant Sciences, ETH Zurich, 8092 Zurich, Switzerland; 2Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf,

Switzerland; 3Agroscope FAL Reckenholz, Swiss Federal Research Station for Agroecology and Agriculture, 8046 Zurich, Switzerland

Summary Author for correspondence: Andreas Lüscher Tel: +41 1377 72 73 Fax: +41 1377 72 01 Email: [email protected] Received: 17 February 2006 Accepted: 25 April 2006

• In grasslands, the loss of structural carbon (C) from nonharvested plant parts is a primary C source for the soil. The amount of input depends not only on the size of structural C pools but also on their loss rates. • In the field, we examined the effects of elevated atmospheric partial pressures of CO2 (pCO2) and nitrogen (N) supply on pool size and rates of structural C loss in stubble and roots of perennial ryegrass (Lolium perenne) by using multiple-pulse labelling and steady-state labelling. • Stubble retained structural C for roughly half the time it was retained in roots. Elevated pCO2 combined with low N supply enlarged the pools of roots and stubble. These conditions also stimulated the rate of structural C loss from stubble and, thus, the amounts available for further transformation. • The potential of multiple-pulse labelling as a field technique is highlighted. The stimulation of structural C loss from stubble by elevated pCO2 at low N provides a missing link between increased C assimilation and low yield response and indicates a potentially higher input of structural C into the soil. Key words: elevated CO2, Lolium perenne (perennial ryegrass), managed grassland, pulse labelling, roots, steady-state labelling, structural carbon loss, stubble. New Phytologist (2006) 172: 117–126 © The Authors (2006). Journal compilation © New Phytologist (2006) doi: 10.1111/j.1469-8137.2006.01796.x

Introduction In grasslands, carbon (C) from the nonharvested plant parts of roots and stubble (between ground and defoliation height) is the major input of C to the soil. C input is a prerequisite for the formation of soil organic matter and thus for C sequestration. Knowledge of the mechanisms and processes of C loss and retention in plants is therefore crucial for understanding plant functioning and C cycling in grassland ecosystems. Grasslands cover c. 40% of the land area world-wide (White et al., 2000), and most are permanent or at least not regularly tilled. Their soils usually have a high content of organic matter, which suggests that grasslands have a high potential to sequester C (Scurlock & Hall, 1998; van Ginkel et al., 1999). Perennial grasses are the dominant species in grasslands in humid temperate

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zones. In these zones, large grassland areas are managed and fertilized to provide high-quality fodder for ruminants. Little is known about the loss of structural C from perennial grassland plants (Thornley, 1998) and its response to atmospheric partial pressure of CO2 (pCO2) and soil fertility. Structural C is considered here to be C that has been converted into structural plant biomass such as cell walls and organelles. Structural C is lost primarily by herbivory and the senescence of plant parts, mainly below defoliation height (roots and stubble). These processes operate on time-scales of months after the assimilation of the photosynthates. By contrast, C is lost rapidly after assimilation of C through respiration, rhizodeposition and the transfer of carbohydrates to mycorrhizas and other symbionts. These losses occur within hours to days (Kuzyakov, 2006) and mainly affect recently fixed photosynthates not incorporated

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into structural components. Here, we focus on longer-term processes affecting structural C because (i) much less is known about the loss of structural C from plants than about the loss of mobile compounds, (ii) plant residues are an important biomass input to soil (Kuzyakov, 2006), and (iii) stabilized compounds in structural biomass are probably more stable in soil and make an important contribution to C sequestration (Six et al., 2001). The loss of structural C in perennial grasses has some interesting features unique to this class of plants. First, perennial grasses form sets of tillers, which are continually renewed by young tillers as the preceding ones become senescent, and this leads to a continuous loss of structural C (Harris et al., 1979). Tillering is a prerequisite for the persistence of perennial grasses because it allows regrowth after defoliation by cutting or grazing. At defoliation, the portion of tillers above cutting height is removed. If the growth meristem of a tiller is removed, as is the case in reproductive tillers, the stubble and roots of that tiller decay. From defoliated tillers with an intact basal meristem, new leaves are expanded and new tillers with the associated adventitious roots are produced. In addition, many small tillers start to senesce before they reach defoliation height and also contribute to the loss of structural C (Suter et al., 2001). Secondly, grass tillers are pseudo-stems of leaf sheaths developed by the production of new leaves within the encircling bases of older leaves. In perennial ryegrass (Lolium perenne L.), only a fixed number of three to four leaves per pseudo-stem are maintained (Davies, 1988). The production of new leaves therefore results in the senescence of older leaves and leads to a continuous loss of structural C. Despite the crucial importance of structural C loss in stubble and roots, most information on the response of intensively managed grasslands to elevated pCO2 is based on the production of biomass above cutting height (yield). However, yield is a poor indicator of the response of the plant to elevated pCO2 (Daepp et al., 2001). For example, in our free-air CO2 enrichment (FACE) experiment which had a duration of 10 years, the average response to elevated pCO2 was 38% for C uptake in leaves of L. perenne (Ainsworth et al., 2003), but −2% for the yield at low N supply (Schneider et al., 2004). These results suggest that elevated pCO2 increased (i) respiration and/or (ii) the allocation of assimilated C to nonharvested plant parts. Indeed, many studies found increased root mass under elevated pCO2 (e.g. Fitter et al., 1996; Hebeisen et al., 1997; Jastrow et al., 2000; Reich et al., 2001), which was often interpreted as increased C allocation to roots. Root mass alone is, however, an invalid indicator of C allocation to the roots, because the C flux (allocation) is the result of the pool size (e.g. root mass) multiplied by the turnover rate of the pod. Thus, the observed increase in root mass may not be the result of an increased C flux to the roots but the result of a reduced turnover rate of roots (i.e. increased life span). A number of studies used minirhizotrons to quantify effects of pCO2 and N on root turnover in grasslands (Fitter et al.,

New Phytologist (2006) 172: 117–126

1996, 1997; Arnone et al., 2000), but they provide only indirect information on C losses. No such studies are available for stubble, with the exception of measurements of leaf longevity by Craine & Reich (2001). Others quantified the C balance of the ecosystem by measuring C exchange (e.g. Aeschlimann et al., 2005) or by C labelling (e.g. Loiseau & Soussana, 1999b), but there is no information on the role of different plant parts in C cycling. In order to shed light on the role of stubble and roots in C fluxes in perennial grass swards, we examine here the rate of structural C loss from these plant parts in L. perenne. Perennial ryegrass is the main grass species of managed grasslands on fertile soil in temperate zones. To understand the effects of resource availability, swards were exposed in the field to different levels of pCO2 and N supply. The loss rate of structural C from plants was quantified using two isotopic methods, multiplepulse labelling (MPL) and steady-state labelling (SSL). MPL is readily applicable in the field, but relies on measures ensuring the homogenous distribution of assimilated label into structural C. SSL takes place over a long period and guarantees a homogeneous label (Kuzyakov, 2006). However, it requires enclosures, which greatly affect growth conditions, or, as in the experiment here, a change in pCO2. We therefore compared results obtained using these two imperfect methods. The aim of this study was to contribute to the mechanistic understanding of how elevated pCO2 and N supply affect C allocation in grassland plants and the subsequent loss of structural C.

Materials and Methods Experimental set-up The field experiment with Lolium perenne L. was conducted on a eutric cambisol (Lüscher et al., 1998) at Eschikon near Zurich, 550 m above sea level. The experimental field consisted of three blocks and has been described in detail by Hebeisen et al. (1997). Each block contained two circular areas of 18 m in diameter, one with 60 Pa pCO2 achieved by FACE and a control area of the same size with ambient pCO2 at around 36 Pa. The two areas were at least 100 m apart to prevent an increase of pCO2. FACE does not require an enclosure and, thus, the microclimate of the experimental area was not altered (Lewin et al., 1992). FACE took place from dawn to dusk during the entire growing season (March to November) if the air temperature was above 5°C. As structural C in stubble and roots was investigated by sequential destructive harvests, swards of L. perenne var. Bastion were grown in large PVC tubes, 0.2 m in diameter and 0.6 m in depth. The tubes were filled with soil and inserted into the field with their edges level with the soil surface and packed tightly together to form a closed sward. The soil was taken from the top 0.2 m of permanent swards of L. perenne in the experimental areas of the appropriate CO2 treatment. In June 1999, six seedlings of L. perenne were transplanted from the

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Table 1 (a) Monthly average air temperatures at 2 m above the ground and monthly sums of precipitation during the measurement period at the experimental site in Eschikon, Switzerland (8°41′ E, 47°27′ N) and (b) accumulated sum of temperature above 0°C (TS) and sums of precipitation for the periods between the destructive harvests of stubble and roots (a) Year

Month

Temperature (°C)

Precipitation (mm)

1999

November December January February March April May June July August September October November December January February March

1.3 0.9 − 0.7 3.5 5.2 9.3 14.4 17.3 16.2 18.1 14.1 9.4 4.8 2.8 0.7 2.8 6.0

108 149 31 137 93 56 93 104 215 111 127 66 76 39 103 43 237

2000

2001

(b) Number

Date1

Accumulated TS (°C)

Precipitation (mm)

1 2 3 4 5

2.11.99 14.03.00 27.06.00 28.10.00 15.03.01

0 323 1262 1800 470

0 453 318 508 418

1

Dates are given as day month year.

glasshouse into each tube, and fertilized with 5.5 g phosphorus (P) m−2 year−1 and 24.1 g potassium (K) m−2 year−1, which was assumed to be nonlimiting for plant growth (Daepp et al., 2000). Monthly average air temperatures and monthly sums of rainfall during the period of measurement are presented in Table 1a. The tubes in each experimental area were divided into two groups of 10; each group received either 14 or 56 g N m−2 year−1. The fertilizer was split into six portions according to the expected biomass production of the plants and applied as follows: 15% at the beginning and 15% in the middle of the first period of regrowth and 20, 20, 15 and 15% at the beginning of each subsequent period of regrowth. In March 2000, after 9 months, the microswards were divided into two sets. Set 1 remained at the original level of pCO2, thus experiencing a steady CO2 treatment. Set 2 was exchanged between the two levels of pCO2: swards that established under ambient pCO2 were moved to elevated pCO2 and swards that established under elevated pCO2 were shifted to ambient pCO2.

Data collection and measurements The measurements were carried out from 2 November 1999 to 15 March 2001. Yield (harvestable biomass above a cutting height of 0.05 m) was determined five times in 2000. Stubble (shoot material below defoliation height) and roots were sampled at five destructive harvests. Dates are given in Table 1b. Stubble was cut at the soil surface, washed and separated into green stubble (mainly pseudo-stems), residual leaf lamina, necrotic material and short pieces of roots that were found close to the tiller base. All plant material was dried at 65°C for 48 h. Harvested tubes were replaced immediately by preestablished swards to form a continuously closed canopy. The soil was sampled to a depth of 0.15 m, where 90–95% of total root mass is found (Hebeisen, 1997). Roots were sampled by washing the soil in two sieves with a mesh size of 2 mm and 250 µm. Roots were separated manually from litter, stubble and soil macro-biota. Organic and mineral fractions were separated by flotation in H2O and dried immediately. For each harvest, the average bulk density of the soil was used to calculate the biomass of roots per m2. Multiple-pulse labelling (MPL) with 14C and 14C analysis The aim of MPL was to distinguish between old and new structural C and to assess C loss from the rate at which old was replaced by new C. This required homogenous labelling of old C and could not be achieved by a single pulse. We therefore labelled swards with three pulses of 14C at 4-wk intervals from August to October 1999 during their establishment. For each pulse, a 0.4-m3 Plexiglas chamber was placed over the tubes and 74 MBq m−2 of 14CO2 was released into the chamber and assimilated by the plants. Two weeks after each labelling, the biomass above cutting height was removed. 14C in above-ground biomass was analysed according to Suter et al. (2002). Briefly, a sample of 20 mg was digested by 4 mg of cellulase and 4 mg of maceroenzyme in 200 µl of phosphate buffer (pH = 6) for 24 h at 45°C, and then by 1 ml of Soluene350 (PerkinElmer, Wellesley, MA, USA) for another 24 h at 45°C. Thereafter, 15 ml of Hionic-Fluor (PerkinElmer) were added and 14C was counted by liquid scintillation (Packard 2500TR; PerkinElmer). Internal standards with a known amount of 14C were used to correct for quenching and the background of 14C was determined from unlabelled material. Samples of 100 mg of roots (> 2000 µm) and 50 mg of fine roots (250–2000 µm) were oxidized in a biological sample oxidizer (OX 400; R. J. Harvey, Hillsdale, NJ, USA) at 900°C for 4 min. Emitted pCO2 was trapped in 15 ml of Oxosol scintillation solution (National Diagnostics, Atlanta, GA, USA) and 14C was counted by liquid scintillation (Wallac 1414 Win Spectral; PerkinElmer). Unlabelled material was used to determine the background of 14C. As there were no

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significant differences in biomass or in 14C values between plants in the two sets under the same pCO2, the data of sets 1 and 2 were pooled. Steady-state labelling (SSL) with

13C

and

13C

analysis

SSL was achieved by exposing plants to a continuously labelled atmosphere over a period of several months and resulted in homogenously labelled biomass. This is difficult to achieve in the field without changing the microclimate using some sort of enclosure. In a new approach, we achieved SSL in the field with the CO2 gas used for FACE. This originated from a fossil source and the δ13C at elevated CO2 was around − 37‰ compared with −29‰ at ambient CO2 (Nitschelm et al., 1997). The swards in set 2 were established over 5 months ( June–November) in the two steady atmospheres with a different 13C signal and were exchanged between these atmospheres in March. The exchange resulted in a 13C label of new C different from the structural C produced before. The substitution rate of old by new C indicated the loss rate of structural C. Because FACE was suspended below 5°C (winter), the steady-state labelling lasted until November under FACE and until March under ambient pCO2.

13C in 2-mg samples of biomass was measured in a continuous-

flow mass spectrometer (PDZ Europa, Sandbach, UK) and the results were expressed in δ units (‰): R  δ13C = 1000 ×  b − 1  Rs 

Eqn 1

(R b, 13C/12C of biomass; R s, 13C/12C of standard Vienna-Pee Dee Belemnite (International Agency of Atomic Energy, Vienna, Austria).) Statistical analyses and calculations Biomass data were analysed using the GLM procedure in SAS (SAS Institute, 1999). The experimental design was a split plot with three blocks (Gomez & Gomez, 1984). pCO2 was the main-plot factor and N fertilization the subplot factor. pCO2 was tested against the interaction pCO2 × block. As pCO2 × block has only two degrees of freedom, the F-test has low power and effects of pCO2 are more readily detected as interactions with the subplot factor. In order to compare the effects of elevated pCO2 and N supply on the loss of structural C in stubble and roots, we estimated loss rates from isotope data. The data suggested an

Fig. 1 14C activity (a, b) and δ13C (c, d) in stubble (a, c) and roots (b, d) of Lolium perenne during the experimental period as affected by two pCO2 treatments (, 36 Pa; , 60 Pa) in combination with two levels of N supply (, 14 g m−2 year −1; , 56 g m−2 year −1). Symbols show means of six replicates in (a) and (b) and of three replicates in (c) and (d), with standard deviations as error bars. Vertical arrows in (a) and (b) indicate the date of the three pulse-labellings with 14 C; horizontal bars in (c) and (d) indicate the duration of the steady-state labelling with 13C.



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exponential decay of 14C and 13C in stubble and roots (Fig. 1). We therefore assumed two pools of structural biomass, one each for roots and for stubble, and calculated first-order models. The data gave no indication of a more complex model and the most parsimonious was used. Parsimonious models with few parameters may be as able to capture dynamics in ecological data as complex models (Schneider et al., 2006). Figure 1 suggested a slowing down of C loss during winter. Therefore, the sum of the daily mean temperatures above 0°C (TS) was used as the time variate. This measure represents time over winter more realistically because it takes into account temperature effects on plant metabolism (West & Brown, 2005). Given this, the model:

with season (Diels et al., 2001), the input source δi was the average δ13C in all yield biomass sampled between two harvests of stubble and roots, calculated for each block and treatment. Effects of pCO2 and N on rates of C loss were analysed in a covariance analysis (ANCOVA) with time as the covariate using the MIXED procedure in SAS (Milliken & Johnson, 2002). In order to account for the split plot design, pCO2 and pCO2 × block were stated as random (Milliken & Johnson, 2002). The method of Kenward–Rogers was used to obtain the correct denominator degrees of freedom (Littell et al., 1996).

A = A0 ⋅ e −k ⋅TS

Size of biomass fractions

Eqn 2

was fitted to the isotopic activity A for each combination of factors and block. A0 is the activity at TS = 0 and k is the rate of C loss, which was recalculated to an annual basis using the annual TS during the experimental period of 3435°C. The isotopic activity A was different for the two labelling methods. For MPL, A was the total 14C activity (Bq m−2) in stubble and roots, in order to account for size variation of biomass pools between individual tubes of the same treatment (Milchunas & Lauenroth, 1992). For 13C SSL, A was the fraction of old C (FOC), calculated as: FOC = 1 −

δt − δ0 δi − δ0

Eqn 3

(δ0 and δt, the δ13C in the biomass at TS = 0 and TS = t, respectively.) As discrimination of 13C in assimilation varied

Results

High N resulted in a 60% stimulation of yield above cutting height compared with low N [analysis of variance (ANOVA): N, P < 0.0001; Table 2]. The response of annual yield to elevated pCO2 was 9% at low N and 21% at high N (ANOVA: pCO2, P < 0.1; pCO2 × N, P < 0.05). Elevated pCO2 increased the pool of residual leaf lamina below cutting height by 50% at low and 26% at high N (ANOVA: pCO2, P < 0.01; Table 2); the pool of residual leaf lamina was larger at low than at high N (ANOVA: N, P < 0.001). The pools of green and necrotic stubble were nonsignificantly larger at elevated than at ambient pCO2; the total pool of stubble was, on average, 21% larger at elevated than at ambient pCO2 (ANOVA: pCO2, P < 0.1; pCO2 × N, not significant). Elevated pCO2 significantly enlarged the pool of coarse roots by 37% on average (ANOVA: pCO2, P < 0.05), independent of N fertilization (ANOVA: pCO2 × N, not significant).

Table 2 Annual dry mass yield and average size of the above- and below-ground plant biomass pools as green stubble, residual leaves (below 0.05 m cutting height), necrotic stubble and coarse and fine roots of Lolium perenne swards as affected by atmospheric pCO2 (36 and 60 Pa pCO2) and N fertilization (14 and 56 g m−2 year −1) Residual biomass (g m−2) Stubble

Roots

Total

N (g m−2 year −1)

pCO2 (Pa)

Yield (g m−2 year−1)

Green

Necrotic

Residual leaf lamina

Sum

Coarse

Fine

Sum

14

36 60 36 60

953 1041 1456 1765

137 189 189 200

224 260 220 264

49 74 40 50

410 522 449 514

159 229 148 194

377 400 385 393

537 628 533 592

950 1152 1000 1108

0.06 0.0001 0.03 6 46.3

ns 0.002 0.04 30 10.3

ns ns ns 30 12.2

0.003 0.0004 ns 30 4.5

0.07 ns ns 30 16.9

0.03 0.03 ns 30 10.2

ns ns ns 30 15.3

0.09 ns ns 30 20.0

0.04 ns ns 30 29.9

56 ANOVA pCO2 N pCO2 × N n SE

P-values of analysis of variance (ANOVA), number of measured tubes (n) and mean standard errors (SE) are shown. ns, not significant.



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The pool of coarse roots was higher at low N than at high N (ANOVA: N, P < 0.05). The pool of fine roots was unaffected by elevated pCO2. The total pool of roots, however, increased by 14% at elevated pCO2 (ANOVA: pCO2, P < 0.1). Hence, elevated pCO2 enlarged the total pool of nonharvested biomass by 21% at low N and by 11% at high N (ANOVA: pCO2, P < 0.05; pCO2 × N, not significant). 14C

activity and δ13C in stubble and roots

The activities were different between the treatments but this was not relevant for our research question, as the rate of change (the slope of the decay curve) and not the absolute values indicated the loss of structural C. In general, the activity of 14C in stubble and roots (Fig. 1a,b) was lower at high N than at low N. The δ13C in stubble increased from around −39 to −30‰ when swards were shifted from elevated to ambient pCO2 (open symbols in Fig. 1c). A shift in the reverse direction (closed symbols) decreased the δ13C from c. −29 to −36‰. At the last sampling in March 2001, the δ13C increased slightly as a result of the suspension of fumigation during winter. Changes in the δ13C in roots (Fig. 1d) were slower than in stubble. Rates of carbon loss In general, there was a good agreement between the two labelling methods. By both methods, rates of C loss from stubble were estimated to be between 2.3 and 3.4 year−1 (Table 3; Fig. 2), and more than double those of roots. Rates of C loss from roots were 1.1–1.2 year−1 when measured by MPL and 0.7– 0.8 year−1 when measured by SSL.

The rate of C loss was higher at low N than at high N when measured by MPL (ANCOVA: N, P < 0.05) and this was confirmed by SSL. MPL revealed that elevated pCO2 stimulated the loss rate from stubble by 33% at low N; SSL also indicated an increased C loss rate for this treatment. MPL showed a 12% reduction of the loss rate from stubble caused by elevated pCO2 at high N, whereas SSL indicated a trend to a higher loss rate for this treatment. Neither N supply nor pCO2 significantly altered the loss rates from roots determined by either method.

Discussion The most striking results of our experiment were that stubble retained structural C for roughly only half the time it was retained in roots (Fig. 1) and that pCO2 and N supply had marked effects on the amount of structural C lost from stubble but not from roots (Fig. 2, Table 3). Two conclusions can be drawn from this: (i) processes operating at different paces are responsible for structural C loss from stubble and roots, and (ii) levels of pCO2 and N supply affect these processes differently. There was a good agreement between the two labelling approaches, highlighting the potential of multiple-pulse labelling as a field technique to assess long-term C loss from plants. Roots retain structural C for a longer time than stubble There are very few investigations on the long-term loss of C from grassland plants, although this process is crucial for C input to the soil and C allocation within grassland plants. Measurements of structural C losses from stubble as a result of biomass

Table 3 Rates of carbon (C) loss per year estimated using multiple-pulse labelling (MPL) and steady-state labelling (SSL) in stubble and roots of Lolium perenne swards at 14 and 56 g N m−2 year −1 combined with two pCO2 treatments (36 Pa ambient pCO2 and 60 Pa elevated pCO2) Stubble

Roots

MPL

SSL

MPL

SSL

N (g N m−2 year −1)

pCO2 (Pa)

year −1

r2

year −1

r2

year −1

r2

year −1

r2

14

36 60 36 60

2.57 3.43 2.59 2.27

0.84 0.82 0.87 0.77

2.38 2.96 2.27 2.40

0.94 0.86 0.85 0.93

1.24 1.18 1.21 1.08

0.69 0.71 0.59 0.52

0.83 0.65 0.76 0.75

0.88 0.91 0.92 0.90

56 pCO2 N pCO2 × N n SE

ns 0.03 0.04 30 0.30

ns ns ns 12/15 0.23

ns ns ns 30 0.21

ns ns ns 12/15 0.10

Data were calculated from single-pool decay models fitted to 14C activities (MPL) and fractions of old C derived from δ13C (SSL) over cumulative temperature sum. Coefficients of determination (r2) of the regressions, number of measured tubes (n) and mean standard errors (SE) of the estimates are shown. All regressions were significant at P < 0.001. ns, not significant.



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Fig. 2 14C activity (a, b) and fraction of old carbon (C) (FOC; c, d) derived from δ13C in stubble (a, c) and roots (b, d) of Lolium perenne swards over cumulative temperature sum (TS) above a threshold temperature of 0°C as affected by atmospheric pCO2 (, 36 Pa; , 60 Pa) and N fertilization (, 14 g m−2 year −1; , 56 g m−2 year −1). Large symbols show means of six replicates in (a) and (b) and of three replicates in (c) and (d); small symbols represent fitted regression lines. Error bars show standard deviations. Loss rates and coefficients of determination of the fitted linear regressions are shown in Table 3.

turnover are particularly scarce and represent a significant gap in the data available for modelling plant growth and C cycles in grasslands (Thornley, 1998). Most studies use C labelling to investigate short-term fluxes of nonstructural C such as respiration, rhizodeposition and transfer to symbionts (Kuzyakov, 2006). Because of the scarcity of studies assessing the loss of structural C, our estimates of structural C loss rates are best compared with the few investigations on the turnover of plant organs. Demographic studies on tillers suggested an average turnover rate of tillers of c. 1.8 year−1 (Bullock et al., 1994). This is understandably lower than our estimated loss rate of c. 2.4 year−1 (Table 3) because the continuous turnover of leaves is not taken into account in the demography of whole tillers. Troughton (1981) measured turnover rates of grass roots of c. 1 year−1, which compare well with our loss rates of between 1.2 and 0.7 year−1 (Table 3). Yang et al. (1998) investigated leaf and root formation in the apical meristem and found that the number of phytomers (apex modules) bearing a root was approximately double the number bearing active leaves. This indicates that twice as many active roots as leaves were present on a tiller. Thus, the turnover rate of roots was half that of leaves and corresponds well to our findings.

The two labelling methods agreed qualitatively and quantitatively and were able to detect significant differences between plant compartments and effects of pCO2 and N supply. Labelling structural C by multiple pulses has therefore the potential to quantify loss of structural C without altering the microclimate by the use of enclosures or changes in pCO2. The measured loss rates suggest considerable differences in structural C turnover between stubble and roots. In grasses, a considerable long-term loss of structural C from stubble originates from the generation and senescence of whole tillers. However, even without tiller death, the continuous production and decay of leaf sheaths result in loss of structural C from stubble and in litter production. These processes are determined by seasonal variations, generative and vegetative development, management and weather (Woodward, 1998). In reproductive growth, the shoot apex which is developing into an inflorescence is lifted above the foliage and the stubble of these tillers senesces after defoliation. Thus, the proportion of reproductive tillers has a large effect on the loss of tillers (Fulkerson & Donaghy, 2001). Shading of tillers during regrowth regulates the density of tillers and causes the senescence of small tillers before they reach defoliation height (Suter et al., 2001). These small tillers



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may not establish their own root system and we hypothesize that their senescence primarily contributes to structural C loss from stubble. Together with the continuous leaf sheath production below cutting height, tiller turnover at least partly accounts for the observed differences in loss rates of structural C between stubble and roots. Another loss of structural C in defoliated grasses results from the movement of C above cutting height caused by the expansion of unfolded leaves existing in the stubble when labelling took place. These losses were quantitatively negligible as the yield of the first regrowth, which started 2 wk after the last 14C pulse was applied, contained only 1.4% of the 14C in the stubble and roots. Losses of 14C in nonstructural C by translocation or exudation must also have been small, as large losses by these means would have caused a sharp decay of the 14C label during the first regrowth examined, which was not found in the data. Nonstructural C losses may, however, be more important in the first few days after the pulse labelling. Contrasting responses of roots and stubble to a changed availability of resources To the best of our knowledge, this is the first experiment allowing the comparison of losses of structural C from roots and stubble, in response to an altered availability of the two primary growth resources, CO2 and N. A few studies have been carried out in permanent managed grasslands in which the effects of elevated pCO2 on the appearance and fate of roots were investigated using minirhizotrons, and most of these studies tended to find increased root turnover under elevated pCO2 (Fitter et al., 1996, 1997; Arnone et al., 2000). Nutrient status (Fitter et al., 1997) and defoliation frequency (Whitehead, 1995) also affect the turnover of biomass in grasslands. Loiseau & Soussana (1999b) are the only researchers to quantify the effects of elevated pCO2 on the loss of C from a total pool of residual biomass. They used 13C steady-state labelling at elevated pCO2 and 13C single-pulse labelling at ambient pCO2. Thus, CO2 effects may be confounded with the methodological effects of the labelling technique. In addition, they did not consider stubble and roots as two separate pools of residual biomass, although the results presented here suggest that stubble and roots differ widely in their C retention and their response to elevated pCO2 and N supply (Table 2). Altered C allocation under elevated pCO2 was suggested by various investigations in swards of L. perenne, grown under similar management conditions in the same FACE array. Leaf photosynthesis was enhanced by elevated pCO2 by c. 38% over several years (Ainsworth et al., 2003). Hence, elevated pCO2 increased the availability of C in source leaves. In a 14C tracer study in the field, Suter et al. (2002) found increased short-term allocation of C to roots at elevated pCO2, consistent with experiments in a controlled environment (Gorissen, 1996; Cotrufo & Gorissen, 1997). Such short-term allocation is, however, attributable to nonstructural C, which in the longer


term may be respired or reallocated to expanding leaves and new tillers shortly after defoliation. In another field experiment in our FACE array, the harvest index (i.e. the ratio between harvestable and residual biomass) during vegetative regrowth was reduced by elevated pCO2 as a result of increased biomass allocated to the residual plant parts of stubble and roots (Daepp et al., 2001). The reduction in harvest index could be alleviated by increasing the rate of N fertilization, indicating that N limitation promoted biomass allocation to nonharvested plant parts. However, pool size is an inappropriate indicator of the amount of C allocated to nonharvested plant parts because increased root and stubble mass may also be the result of increased life span (reduced turnover rate) at the same amount of C allocation. Consistent with other studies (Loiseau & Soussana, 1999a; Daepp et al., 2001), we observed an increase in the mass of stubble and roots under elevated pCO2, especially at low N. However, this is the only study showing rates of structural C loss to remain unchanged or even to increase at elevated pCO2. These two results together are evidence that C allocation to nonharvested plant parts was increased at elevated pCO2. This may be caused by higher investments of photosynthates in roots in order to maximize the capture of this most limiting resource (Chapin et al., 2002) or by an N limitation of the sink activity of leaves (Fischer et al., 1997). The observed increased loss of structural C from stubble at low N and elevated pCO2 (Table 2) might be a result of stimulated tillering, increased tiller mass or increased leaf turnover. Suter et al. (2002) grew L. perenne under similar conditions in our FACE array and found that tillers shorter than 0.05 m were increasingly produced in the stubble layer under elevated pCO2 at the beginning of regrowth and were reduced towards the end. The size of individual tillers was not altered by elevated pCO2 in this experiment. Evidence of CO2 effects on leaf turnover in L. perenne is largely missing. Craine & Reich (2001) investigated a number of grasses in more extensively managed swards and found that elevated pCO2 generally increased leaf longevity whereas N supply decreased it. In our experiment, the observed 50% increase in the mass of leaf lamina at elevated pCO2 (Table 3) is so pronounced that even slightly reduced loss rates would still result in higher structural C losses. To conclude, it seems most likely that the observed higher C loss rate from stubble at limited N supply and elevated pCO2 was the result of a stimulated tiller turnover. Our conclusion agrees with that of Fulkerson & Donaghy (2001), who hypothesized that after defoliation the production of tillers and the restoration of photosynthetic capacity have a higher priority for the plant than the production of roots. Stubble is important for the response of intensively managed grasslands to elevated pCO2 Our results highlight the crucial, but often overlooked, importance of stubble in the response of intensively managed grasslands


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to global change, especially at limited N supply. Stubble pools showed a much more pronounced reaction to elevated pCO2 than the commonly investigated harvestable biomass above cutting height. Elevated pCO2 stimulated yield production by 9% at low N (Table 2). This is in the upper part of a range of responses between −11 and 10% we observed over 10 years (Schneider et al., 2004). By contrast, stubble pools increased by 26% at elevated pCO2 on average (Table 2). Multiplying this by the associated higher loss rates (up to 33% at low N) makes it clear that biomass production in these pools was much larger. Hence, the increased allocation of C to stubble may at least partly explain the discrepancy between C assimilation (Ainsworth et al., 2003) and yield (Schneider et al., 2004). Stubble biomass may consist of up to 35% nonstructural carbohydrates (Fulkerson & Donaghy, 2001), which were not measured in our study. Nevertheless, even if only the remaining 65% structural biomass is considered, our study still demonstrates that a large amount of biomass production is needed to steadily replace the amount of structural C lost from stubble. Stubble mass was up to half of the yield but its structural C was replaced up to 3.5 times per year (in the low N, elevated pCO2 treatment). Thus, stubble alone may account for more biomass production than yield. The increased productivity and decay of nonharvested plant parts below cutting height means that elevated pCO2 probably increases litter production, especially of litter in the stubble layer. The potential implications of this pCO2-induced shift are manifold. On the one hand, coverage of the soil surface and food availability to soil biota may increase. On the other hand, the litter produced at elevated pCO2 had a lower N concentration (data not shown), probably reducing the decomposability of this litter (Chevallier et al., 2006). Too little is known about most processes involved in litter production and decomposition in grasslands to predict long-term pCO2 effects (van Groeningen et al., 2006). Here, we focused on the fate of nonharvested plant biomass as the first step in the long chain of C sequestration into the soil. However, measurements of ecosystem CO2 exchange may provide an integrated view of C turnover in the ecosystem. In the L. perenne swards in our FACE experiment, Aeschlimann et al. (2005) measured increased respiration at night under elevated pCO2, and this increase was more pronounced at low N than at high N fertilization. The estimated annual net C uptake was also highest at elevated pCO2 and low N. This agrees well with our measurements suggesting that such stimulated ecosystem C uptake may be a result of increased loss of structural C from stubble, demonstrates impressively the importance of this often overlooked plant part for the allocation and loss of biomass in grasses. Conclusions Our investigation has revealed distinct differences among yield, stubble and roots in response to a change in the availability

of resources, as induced by elevated pCO2 and N supply. The two labelling techniques used to measure C loss from stubble and roots agreed well and suggested, at elevated pCO2, an increased loss of structural C from stubble when N supply was limited. This highlights stubble as a link between strongly increased photosynthetic C uptake and weak yield response under elevated pCO2 and as an important, but often overlooked, source of C input into soil.

Acknowledgements We acknowledge the technical support of U. Aeschlimann, A. Dürsteler, V. Escalante, T. Flura, V. Hebeisen, C. Marshall, R. Muheim and G. Williams. Comments by U. Aeschlimann, P. Hill, L. Limer and three anonymous reviewers considerably improved the manuscript. We would like to thank K. Girgenrath (Laboratory of Organic Chemistry, ETH Zürich) for counting 14C samples and H. Roth for statistical advice. This research was funded by ETH Zurich and COST (Action 627).

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