Decomposition of
13
C-labelled wheat root systems following
growth at dierent CO
2
concentrations
Margret M.I. Van Vuuren
a, b, David Robinson
b,*, Charles M. Scrimgeour
b, John
A. Raven
a, Alastair H. Fitter
caDepartment of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK bScottish Crop Research Institute, Dundee DD2 5DA, UK
cDepartment of Biology, University of York, P.O. Box 373, York YO10 5YW, UK
Received 19 April 1999; received in revised form 31 August 1999; accepted 20 September 1999
Abstract
We tested whether the amounts of carbon (C) mineralized from decomposing wheat (Triticum aestivum L. cv. Tonic) roots were related to the quantity (i.e. root dry weight per plant) or the chemical composition of material which had been grown at ambient or elevated CO2concentrations (350 or 700 mmol CO2 molÿ1). Plants were grown in 13C-depleted CO2 to distinguish
root-derived C from soil-derived C. Over periods of up to ca. 400 d, root C, soil C and nitrogen (N) mineralization were measured from: (i) root systems left in situ in soil; (ii) soil after removal of visible roots; and (iii) equal amounts of roots added to fresh soil. Root systems in situ showed transiently faster C mineralization rates after growth at elevated [CO2] compared with
ambient [CO2]. Ultimately, there were no [CO2]-related dierences in the amounts of C or N mineralized from root systems in
situ. Speci®c rates of C loss from extracted roots were not signi®cantly dierent for roots from the two [CO2] treatments. The
potential accuracy of the13C method was demonstrated and13C/12C fractionation during root decomposition was negligible. We conclude that when wheat is grown under elevated [CO2], subsequent root decomposition will not necessarily be aected. If it
does, it is likely to do so via an eect of [CO2] on the amounts of root material produced per unit of soil rather than on the
chemical quality of that material.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Decomposition; Elevated CO2; Root system;Triticum aestivum; Wheat;13C
1. Introduction
Plant biomass production usually increases when at-mospheric carbon dioxide concentrations ([CO2]) are
elevated and its chemical composition may dier from that produced at current ambient [CO2] (e.g. Conroy
and Hocking, 1993; Owensby et al., 1993; Jackson et al., 1994). These eects depend on plant species and interactions with other environmental factors, e.g., water and nutrient availability, temperature. Likewise, eects of [CO2] on litter decomposition and nutrient
mineralization rates depend on changes in organic matter quantity and quality, soil biota and interactions with physicochemical conditions in the soil (O'Neill and Norby, 1996; Arp et al., 1997; Heal et al., 1997).
Inputs of plant organic matter to soils occur conti-nually, but at variable rates, during the life of a plant, i.e. as above- and belowground plant parts, leachates and exudates. Large, discrete inputs occur when indi-vidual plants die. For annual crops, such inputs Ð particularly of roots Ð occur at harvest and may con-tribute nutrients to subsequent crops.
Only in a few studies (e.g. Swinnen et al., 1995) has in situ root decomposition of annual crops been inves-tigated and none in relation to [CO2]. Information is
also lacking about how [CO2]-induced changes in root
quality (i.e. chemical composition) and quantity
in¯u-0038-0717/00/$ - see front matter#2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 7 4 - 1
www.elsevier.com/locate/soilbio
* Corresponding author. Tel.: +44-1382-562-731, ext. 2530; fax: +44-1382-562-426.
ence their subsequent decomposition. If elevated [CO2]
increases C-to-nutrient ratios of root debris, decompo-sition may be slower.
Information about the eects of elevated [CO2] on
root decomposition has been obtained on homogenized root subsamples, using grasses (Gorissen et al., 1995; Van Ginkel et al., 1996; Franck et al., 1997; Robinson et al., 1997), cotton (Torbert et al., 1995) and tree seedlings (Cotrufo and Ineson, 1995). In a study by Van Ginkel and Gorissen (1998), the in situ decompo-sition of 14C labelled perennial ryegrass (Lolium per-enne L.) roots was measured. Often, studies of root decomposition have involved material sampled from young, non-senescent plants rather than naturally senescent material. This is a particularly important issue when considering C loss from the roots of annual species (e.g. cereals). These exhibit little root turnover pre-anthesis (Van Vuuren et al., 1997), most root mor-tality occurring synchronously at the end of the indi-vidual plant's life. This pattern contrasts markedly with the continual root turnover seen in perennials (Fitter et al., 1996).
Here, we report experiments designed to test
whether the amounts of C mineralized from decompos-ing wheat roots were related to the quantity (i.e. root dry weight per plant) or the chemical composition of root systems grown at ambient or elevated [CO2] (350
or 700 mmol CO2 molÿ1). We compared C
mineraliz-ation from root systems in situ (in which the amount of root material in the soil was dependent on the pre-vious growth of the plants under dierent [CO2]) with
that from equal weights of root material added to soil. We used 13C-labelling to distinguish root-derived C from soil-derived C, which also aorded an opportu-nity to test that technique's accuracy in decomposition studies.
2. Materials and methods
2.1. Soil and roots used in mineralization experiments
Soil and root samples were obtained from the exper-iment described by Van Vuuren et al. (1997); see Table 1. Brie¯y, spring wheat (Triticum aestivum L. cv. Tonic) was grown at 350 or 700 mmol CO2 molÿ1
(ambient and elevated [CO2] treatments, respectively)
and with frequent or infrequent watering (`wet' and `dry' treatments, respectively), in a factorial exper-iment. Samples from only the `wet' treatments were used here. Plants were grown in long vertical contain-ers (120 2.5 5 cm) with removable front covers, allowing access to the soil and the growing root sys-tem. Each container was ®lled with sandyloam soil (1.35 kg at 1058C; sieved (2 mm); 3% total C, 0.21% total N, 0.13% total P and 0.49% total K), of pH 6.1 and fertilized with N, P and K.
The CO2 to which the plants were exposed was
depleted in 13C (see Gordon et al., 1995), allowing C derived from the wheat to be distinguished from that present in the soil: see Table 1). In elevated [CO2],
plants were exposed to CO2 more 13C-depleted than Table 1
Design of the [CO2] and wheat growth experiment of Van Vuuren et al. (1997) from which soil and root samples were obtained for the present series of experiments. Plants were grown under one of two watering regimes (`wet' or `dry'), but samples from only the `wet' treatments were used here. Abbreviations: n: no; y: yes; +: used;ÿ: not used; n.d.: not determined
[CO2] during plant growth (mmol CO2molÿ1)
350 700 350 700
Design of Van Vuuren et al.'s (1997) experiment (`wet' treatment)
Number of plants at ®nal harvest (116 d growth) 4 3a 4 4
Root video recording n n y y
Extraction of roots y y n n
Measurement of soil inorganic N at ®nal harvest y y n n
Samples used for decomposition experiments
Series (i) Soil with root systems in situ ÿ ÿ + +
Series (ii) Soil from which root systems were removed + + ÿ ÿ
Series (iii) Soil to which equal amounts of roots were added + + ÿ ÿ Amounts and composition of extracted roots (mean values, S.E. in parenthesis)
Root dry weight per plant (g) 1.31 (0.05) 1.42 (0.08) n.d. n.d.
Root [C] (mg gÿ1dry weight) 418 (1) 432 (3) n.d. n.d.
Root C per plant (mg) 549 (23) 611 (30) n.d. n.d.
Rootd13C (-) ÿ38.3 (0.1) ÿ41.1 (0.1) n.d. n.d.
Root [N] (mg gÿ1dry weight) 24.1 (0.9) 24.1 (2.7) n.d. n.d.
Root N per plant (mg) 32 (1) 34 (5) n.d. n.d.
Root C-to-N (mass ratio) 17 18 n.d. n.d.
were those grown at ambient [CO2]. Plants were grown
for 116 d, at which point they were in the ripening stage (Large, 1954), although not yet completely senesced.
Certain data in the decomposition experiments described below are quoted as `per plant'. This means that those amounts of C or N were calculated for that amount of soil which supported one wheat plant in the Van Vuuren et al. (1997) experiment. Throughout, all data are quoted as means2S.E.
2.2. C and N mineralization experiments
Three series of experiments were performed.
2.2.1. Soil with root systems in situ, series (i)
This series was designed to measure the decompo-sition of root systems left in situ following the harvest-ing of shoots after 116 d growth (Table 1), simulatharvest-ing the fate of wheat roots in the ®eld when soil is left unploughed at the end of the growing season. These root systems were present in containers which had been used for nondestructive video recordings of root growth in the Van Vuuren et al. (1997) experiment (Table 1).
Decomposition measurements began on the day that shoots were harvested (5 December 1994). Four repli-cate containers per [CO2] treatment were watered to
0.22 g water gÿ1soil and their front covers replaced by ®ne nylon mesh. Each container was placed horizon-tally in a PVC pipe (1.3 m long, 64 mm dia) in an incubator room at 148C (minimum 128C, maximum 168C) in darkness, after taking gas samples from the pipe via two gas sampling ports 34 cm from either end. The pipes were closed with rubber bungs, leaving a 1.25m inner pipe length and an estimated `head space' of 2750 cm3in each. At the ®nal harvest (116 d: Table 1), concentrations of inorganic N with soil depth were measured by Van Vuuren et al. (1997); these were taken as the amounts of inorganic N pre-sent at the start of the in situ decomposition exper-iment.
The containers were incubated for 29 sequential periods, ranging from 3 d at the start of the exper-iment to 42 d at the end, over a total of 404 d. At the end of each period, duplicate gas samples were taken from each pipe to measure [CO2]. For 17 of these
periods, two other gas samples were taken from each pipe for determination ofd13C in CO2. After sampling, each container was removed brie¯y from its pipe. Each pipe was ¯ushed with outside air and one gas sample taken for background measurement of [CO2]. If
necess-ary, moisture losses from each container were replaced with distilled water (always <10 g) and any conden-sation on the inside of the pipe removed.
After 404 d, each soil column was cut into 11 layers.
Visible root fragments were collected manually from each and bulked for each plant. Roots were rinsed with water, ovendried (608C, 24 h) and weighed. Root [C], [N] and d13C were determined as described in Sec-tion 2.3. Soil moisture contents and inorganic N con-centrations were measured as described by Van Vuuren et al. (1997).
2.2.2. Soil from which root systems were removed, series (ii)
This series was designed to detect the presence of root-derived C in soil from which roots grown at dierent [CO2] had been removed. Such C might be
expected to be in the form of detached root fragments, soluble and insoluble rhizodeposits and in microbes and microbial metabolites.
After 116 d growth (Table 1), visible roots were extracted manually from soil and the soil retained for incubations. (Extracted roots were washed with water, dried (608C, 24 h) and used in Series (iii) below; Table 1). The soil from each container was mixed, triplicate subsamples (75 g wet weight) weighed into separate 125 cm3 gas-tight glass jars and a sample dried at 1058C to measure its moisture content (9 December 1994). The soil-containing jars and three soil-free jars were closed with rubber bungs ®tted with gas sampling ports. The average headspace volume of each jar was 63 cm3.
Jars were incubated for 20 sequential periods lasting from 6 to 33 d and 401 d in total. At the end of each period, two gas samples were taken from each jar for measurements of [CO2] and d13C, respectively.
Head-spaces were ¯ushed with outside air and [CO2]
measured in soil-free jars. At the end of the ®rst period, soil moisture contents, which varied between 0.16 and 0.18, were adjusted to 0.22 g water gÿ1soil.
At the end of the experiment, 7 g subsamples of soil from each triplicate jar were combined and soil inor-ganic N concentrations determined in the composite sample. The soil remaining in each jar was dried to measure its moisture content.
The soil-only control was replicated six times. Each replicate comprised three subsamples of 75 g moist soil (0.22 g water gÿ1 dry soil) in separate 100 cm3 jars. Three additional jars were soil-free. [CO2] andd13C of
CO2 were measured at the end of 14 sequential
incu-bation periods, totalling 466 d. Incuincu-bation of controls began on 11 August 1994.
pre-sented. The incubation of soil from which visible roots were removed was aected only during the ®rst month.
2.2.3. Soil to which equal amounts of roots were added, series (iii)
This series was designed to measure speci®c rates of C loss (i.e. C loss gÿ1 initial root dry weight) from roots grown at dierent [CO2] to test whether those
treatments led to innate dierences in root decomposi-bility, re¯ecting possible dierences in chemical qual-ity.
Equal dry weights of roots obtained previously (see Series (ii) above; Table 1) were added to fresh soil. The roots had been dried at 608C (24 h) and stored at room temperature before being clipped ®nely (<0.5 cm) and redried at 608C. For each plant which had been grown under each [CO2], triplicate 200 mg
sub-samples of root material were mixed with 75 g soil (0.22 g water gÿ1 dry soil) in separate 0.5 dm3 gas-tight jars; one additional jar was soil-free (5 June 1995). Two replicate soil samples were used as soil-only controls. For each soil sample, triplicate 75 g sub-samples of moist soil (0.22 g water gÿ1 dry soil) were weighed in separate 0.5 dm3 jars. One additional jar was soil-free.
All jars were incubated for a total of 303 d in the 148C incubator room mentioned above. Jars contain-ing roots were incubated for 19 sequential periods, ranging from 3 to 37 d. [CO2] andd13C were measured
at the end of 19 and 15 periods, respectively. Soil-only controls were incubated for eight sequential periods ranging from 22 to 54 d; [CO2] was also measured
within incubation periods. Before taking a gas sample, air in the jar was mixed by pumping it in and out of a 25 cm3 syringe. After ¯ushing with outside air, [CO2]
was measured in the jar without soil.
At the end of the experiment, inorganic N concen-trations were determined in one soil sample per jar, the remaining soil was dried to measure its moisture content.
2.3. Chemical and isotopic analyses
Gas samples for [CO2] measurement were taken
using 2 cm3glass airtight syringes. The sample volume was reduced to 1 cm3 (at atmospheric pressure) just before injection into a Hewlett-Packard 5890A gas chromatograph. CO2 gas standards (10 or 30 mmol
molÿ1=1 or 3% v/v), sampled and injected in the same way, were used to calibrate the gas chromato-graph for each sample run. [CO2] was smaller than 30
mmol molÿ1 in about 90% of all gas samples ana-lysed.
Before taking gas samples for d13C analyses, [CO2] data were used to calculate the volume of gas needed
for sampling ca. 100ml CO2(=4.2 mmol CO2at 148C
and 101.3 kPa) This volume, up to a maximum of 12 cm3, was sampled from the head space using a 25 cm3 syringe and immediately transferred to a 12 cm3 evacu-ated glass vial (`Exetainer', PDZ Europa, Crewe, UK). Exetainers were stored at room temperature untild13C analysis. d13C was not determined in CO2 from jars without soil. If CO2 in the sample was <50 ml, d13C
data were discarded as being potentially unreliable. Total C and N concentrations and d13C in root samples were determined by continuous-¯ow isotope ratio mass spectrometry (CF-IRMS; Tracermass, PDZ Europa, Crewe, UK). d13C in gas samples was deter-mined using CF-IRMS with an automated gas-sampling and puri®cation system (Roboprep-G Tracer-mass, PDZ Europa, Crewe, UK). d13C values were expressed in parts per thousand (-), by reference to
the international standard V-PDB:
d13C RsampleÿRstandard=Rstandard 103 1
where R13C=12C: Working standards calibrated to V-PDB were of 200 ml CO2 (=8.5 mmol CO2 at 148C
and 101.3 kPa). In each mass spectrometer run, samples of 50, 100 and 200 ml CO2 of two CO2
stan-dards were included d13C ÿ42, ÿ33or ÿ25-).
Over the range 50±200 ml CO2, d13C varied linearly
with the volume of each CO2standard with a common
slope, g. Sample d13C values (d13Csample) were cor-rected for the eect of sample volume (vsample, ml) by
calculating the d13C value (d13C200) that a sample volume of 200 ml would have produced, according to:
d13C200d13Csample 200ÿvsampleg 2
2.4. Calculations of C mineralization rates
The amount of CO2produced per jar or pipe (=net
[CO2]) and per incubation period was calculated by
subtracting the mean [CO2] of outside air from sample
[CO2] at the end of the period. Net [CO2] was averaged
for the three replicate jars per plant. Net [CO2] data
were converted to mg C mineralized per jar (or pipe) (=Ctotal), according to:
Ctotal 0:01CO2hvm=vm 3
where [CO2]=% CO2 v/v, hv=head space volume of jar or pipe (cm3), vm=volume per mmol CO2 at 148C
(23.6 cm3) and m= mass of C per mmol C (i.e., 12 mg).
Ctotalwas partitioned into C mineralized from wheat
roots (=Croots, mg) and C mineralized from native soil
organic matter (SOM; =Csom, mg) using the following
CrootsCtotal cÿa= bÿa 4
and
CsomCtotalÿCroots 5
where a=d13C of CO2 mineralized from SOM,
b=d13C of roots extracted from soil and c=d13C of CO2 mineralized from soil containing roots.
13
C/12C fractionation during the decomposition of root C was not taken into account and no correction was made
for ambient air CO2 introduced during ¯ushing of jars
with outside air. For incubation periods without measurements of d13C, the parameter c was obtained by linear interpolation between d13C measurements made during the previous and following incubation periods.
2.5. Statistical analyses
For the incubation experiments of soil plus or minus roots in jars (Series (ii) and (iii)), statistical analyses were done on mean values of the triplicate measure-ments per plant or per soil sample. Analysis of var-iance procedures in Genstat 5 (Genstat 5 Committee, 1993) were used for data that were not repeated over time (e.g. C mineralization per sampling day, total inorganic N per plant or per jar).
Amounts of C mineralized were repeated measure-ments for each of the original plants. The appropriate statistical analysis for such data was described in detail by Van Vuuren et al. (1997). Brie¯y, for each plant, data were summarized across time by ®tting an orthog-onal polynomial function of time using Genstat 5. The
estimated parameters of this function were not
repeated measurements and, for a given plant, the par-ameter estimates were uncorrelated. Eects of the [CO2] treatments on each parameter were analysed
using analysis of variance as before.
3. Results
3.1. Soil with root systems in situ (Series (i))
The amounts of C mineralized varied more among replicates of soil with roots grown at elevated [CO2]
than among replicates with roots grown at ambient [CO2] (Fig. 1a,b). For both treatments, C
mineraliz-ation rates decreased with time.
d13C values of total C mineralized were initially more negative in the elevated [CO2] treatment (Fig.
1c,d) because plants in that treatment were more 13 C-depleted (see Materials and methods; Table 1) and, in-itially, more root C was mineralized in that treatment
Fig. 1. C mineralization in soil containing root systems in situ (Series (i): Table 1). (a,b): Total amounts of C mineralized (mg per plant); (c,d):d13C values (-) of mineralized C. (e,f): Amounts of C minera-lized (mg per plant) from soil organic matter (SOM; open symbols) and from roots (closed symbols). Symbols are replicate measure-ments; lines pass through the means. Graphs (a,c,e) are for plants grown at 350mmol CO2molÿ1; (b,d,f) are for plants grown at 700
mmol CO2molÿ1(see Table 1).
Table 2
Net C and N mineralization (mg per plant) in incubations of soil with root systems in situ (after 404 d; Series (i): Table 1) and soil after removal of root systems (after 401 d; Series (ii): Table 1). The origin of mineralized C, i.e. from roots (Croots) and from soil organic matter (Csom), was determined fromd13C values (see text). Roots were from plants grown at 350 or 700mmol CO2mol1: see Table 1. Data are means with S.E. in parentheses
[CO2] during plant growth (mmol molÿ1) Soil with root systems in situ (Series (i)) Soil after removal of root systems (Series (ii))
Croots Csom N Croots Csom N
350 405 (19) 339 (15) 97 (8) 87 (6) 205 (11) 61 (6)
(see below). Meand13C values of extracted roots were
ÿ38.3 and ÿ41.1-for ambient and elevated [CO2],
re-spectively (Table1). As more of the root C decom-posed, d13C values of CO2 became less negative and closer to that of CO2 evolved from soil-only controls
d13C ÿ26:3-).
After 404 d, estimates of C mineralized from root systems grown at ambient and elevated [CO2] were 405
219 and 480253 mg per plant, respectively; the cor-responding amounts of C mineralized from SOM were 339215 and 392221 mg (Fig. 1e,f; Table 2). Separate ANOVAs for each sampling day indicated that signi®-cantly (P< 0.05) more C was mineralized from roots grown at elevated [CO2] after 8, 11, 14 and 17 d (P<
0.1 up to 36 d). However, the accumulated amounts of C mineralized from roots grown at dierent [CO2]
became more similar with time and the analysis of the complete data set indicated no statistically signi®cant dierence between [CO2] treatments. C mineralized
from SOM also did not dier signi®cantly between [CO2] treatments.
At the start of decomposition, C was signi®cantly
P0:005 more concentrated in the dry matter of roots grown at elevated [CO2], although the total
amounts of root C produced under dierent [CO2]
were not dierent (Table1; P0:13). After 404 d,
only 96220 and 8024 mg root C per plant remained in roots recovered from the soil. These data indicate that, respectively, 83 and 87% of ambient and elevated [CO2] grown roots had decomposed during that time.
The corresponding fractions estimated from measure-ments of evolved CO2and itsd13C were 74 and 79%.
Thed13C values of roots recovered at the end of the decomposition experiment were ÿ38.220.4 and ÿ40.7 20.2- for ambient and elevated [CO2], respectively,
statistically indistinguishable (P> 0.1) from those at the start (Table 1).
After 404 d, soil which had supported plants grown at ambient and elevated [CO2] contained, respectively,
12828 and 12124 mg inorganic N per plant. Inor-ganic N was most concentrated in the upper soil layers (Fig. 2), corresponding to the greater root dry weights per unit soil volume at those depths (Van Vuuren et al., 1997). These amounts of soil inorganic N were
Fig. 2. Inorganic N concentrations (mg kgÿ1dry soil) in soil contain-ing root systems in situ (Series (ii): Table 1). Concentrations are shown at the start (0 d; open symbols) of the root decomposition ex-periment and at the end (404 d; solid symbols). Symbols are replicate measurements; lines pass through the means. Graph (a) is for plants grown at 350mmol CO2molÿ1; (b), is for plants grown at 700mmol CO2molÿ1(see Table 1). Total amounts of soil inorganic N (areas under curves, mg per plant) were: (a) 31211 (start), 12828 (end); (b) 32216 (start), 12124 (end).
substantially greater than those present at the start of decomposition, i.e., 31211 and 32216 mg per plant in ambient and elevated [CO2] treatments, respectively
(Van Vuuren et al., 1997). Net soil N mineralization during root decomposition did not dier signi®cantly
P0:474 between [CO2] treatments, ca. 94 mg per
plant (Table 2).
3.2. Soil from which root systems were removed (Series (ii))
d13C values used for partitioning C mineralized into that originating from roots and SOM were obtained from soil incubated after removal of root systems (data not shown), extracted roots (Table1) and CO2
evolved from soil-only controls d13C26:3-). About
85 mg per plant of root-derived C were mineralized from soil after the removal of visible roots during 401 d, irrespective of [CO2] treatment (Table 2). The total
amounts of C and N mineralized from roots or SOM did not dier signi®cantly between [CO2] treatments
(Table 2). At the end of the incubation, total soil inor-ganic N was equivalent to 9227 and 9729 mg N per plant in ambient and elevated [CO2], respectively. Net
N mineralization was ca. 63 mg N per plant in both treatments (Table 2).
3.3. Soil to which equal amounts of roots had been added (Series (iii))
The amounts of C mineralized from fresh soil to which equal amounts of roots had been added were 71 20.6 and 6920.5 mg per jar after 303 d for roots grown at 350 and 700 mmol CO2 molÿ1, respectively
(Fig. 3a,b). Subtracting from these values the average amount of C mineralized in soil-only controls indi-cated that about 40 mg root C were mineralized per jar during 303 d, i.e. about 45% of the added root C.
Root C mineralization was also calculated using d13C values obtained from roots, CO2 evolved from soil with roots added and CO2 evolved from soil-only
controls (Fig. 3c,d; the mean d13C value of ÿ26.4
-was used for soil-only controls). These calculations indicated that 37.820.4 and 37.120.9 mg of root C were mineralized from ambient and elevated [CO2]
treatments, respectively, i.e. amounts similar to those
obtained from direct measurements of CO2
pro-duction. The smaller amounts of root C mineralized compared with the in situ incubations (Fig. 1) were probably because of the establishment of larger mi-crobial populations in the soil during plant growth, an eect absent from the incubations in jars.
At the end of incubation, total soil inorganic N did not dier signi®cantly P0:194 between soils with roots grown at dierent [CO2], being 4.920.1 and 4.4
20.4 mg N for the ambient and elevated [CO2]
treat-ments, respectively. Soil-only controls contained 3.42 0.2 mg N at the end of incubation.
4. Discussion
4.1. Quality vs. quantity of decomposing wheat roots
Initially, more C was mineralized in situ from root systems grown at elevated [CO2] compared with those
grown at ambient [CO2] (Fig. 1), but this eect was
transient. It may have been caused by greater initial concentrations of easily decomposable, non-structural C compounds in roots grown at elevated [CO2] (e.g.
Jongen et al., 1995).
[CO2] had only a small eect on the total amounts
of C mineralized in situ, as it had on root dry weight in the frequently watered treatments of the Van Vuu-ren et al. (1997) experiment (which supplied the ma-terial used in this study: Table 1). When watering was infrequent, however, Van Vuuren et al. (1997) found a 27% increase in root dry weight per plant at elevated [CO2]. Had root systems from the infrequently watered
treatments been available for the decomposition stu-dies, this large dierence in their initial dry weights might have produced greater eects of [CO2] on total
C mineralization than we observed. The eects of [CO2] on the percentage of roots and root-derived C
mineralized in situ may be small. For example, Van Ginkel and Gorissen (1998) found that 5224% of in-itial C was mineralized from ryegrass roots over 230 d irrespective of [CO2] or N treatment. But the initial C
content of the 14C-labelled soil used by Van Ginkel and Gorissen was 18 to 39% greater at elevated than at ambient [CO2], resulting in a 10 to 26% greater
total C mineralization at elevated than at ambient [CO2].
There was little eect of [CO2] on the chemical
com-position of wheat roots in the Van Vuuren et al. (1997) experiment, whether the plants were well watered (Table 1) or not (M.M.I. van Vuuren, unpub data). That was re¯ected in the similarity of the speci®c rates of C loss measured on extracted roots irrespective of previous [CO2] treatment (Fig. 3). Some
non-structural C compounds may have leached from the extracted roots during rinsing before the start of the decomposition experiment (cf. Cotrufo and Goris-sen, 1997), perhaps explaining the absence of the in-itially rapid C mineralization from elevated [CO2]
grown roots.
Our data suggest that elevated [CO2] will not
necess-arily aect subsequent C cycling from arable crop resi-dues in soil. When there is such an eect on wheat, it is more likely to arise from dierent amounts of root per unit of soil rather than from [CO2]-induced
How-ever, even though we incubated root systems from senesced plants in situ, our experimental setup could not mimic ®eld conditions precisely. There, more types of soil biota are present than in the sieved soil used in our experiments (Jones et al., 1998) and soil biota, nutrient availability, temperature and moisture con-ditions ¯uctuate in space and time. Moreover, during the decomposition experiments, our soils were without living vegetation exposed to ambient or elevated [CO2].
The latter probably would have resulted in greater soil moisture contents at elevated than at ambient [CO2]
caused by smaller water use by plants growing at elev-ated [CO2] (Jackson et al., 1994; Hungate et al., 1997a;
Van Vuuren et al., 1997; Arnone and Bohlen, 1998; Nicklaus et al., 1998), thereby aecting soil biota and decomposition. Griths et al. (1998) could detect no eect of [CO2] on the composition of the soil microbial
community from an experiment involving the same wheat cultivar, soil type and growth conditions used by Van Vuuren et al. (1997). Our ®nding of no strong eects of [CO2] on subsequent decomposition of wheat
roots suggests little in¯uence of [CO2] on the
function-ing of that community in terms of C or N mineraliz-ation.
Previous experiments showed no dierence (Cotrufo and Ineson, 1995; Torbert et al., 1995; Robinson et al., 1997), slower (Cotrufo and Ineson, 1995; Gorissen et al., 1995; Van Ginkel et al., 1996), or faster (Robinson et al., 1997) rates of decomposition of roots grown at elevated [CO2] compared with those grown at ambient
[CO2]. In the experiments by Cotrufo and Ineson
(1995) and Franck et al. (1997), the direction and mag-nitude of eects of elevated [CO2] on root
decompo-sition depended on species and, in the experiment by Robinson et al. (1997), on environments. Root quality and decomposition were measured on roots from senesced plants in the experiments of Torbert et al. (1995), Franck et al. (1997) and Robinson et al. (1997); in the others, root `litter' was obtained by sampling roots from rather young plants.
Eects of [CO2] on decomposition have been
investi-gated more often on aboveground plant parts than on roots but, again, few experiments (e.g. Kemp et al., 1994; Akin et al., 1995; Franck et al., 1997) have used naturally senesced material. Though it is often assumed that elevated [CO2] reduces nutrient
concen-trations and nutrient to carbon ratios in litter, evidence for this generalization is still inconclusive (Arp et al., 1997; c.f. Ball, 1997). Likewise, there is insucient evi-dence to conclude that plants grown in elevated [CO2]
produce more recalcitrant litter which decomposes more slowly (O'Neill and Norby, 1996; Arp et al., 1997; Norby and Cotrufo, 1998).
Our work shows that small amounts of root C deposited in soil (relative to the amounts of organic C already present in the soil) can make a
disproportio-nately large contribution to mineralized C. Wheat root systems in situ (Series (i)), including rhizodeposits and root-derived microbial products, were only a small fraction of total soil organic C, yet their contribution of root C to mineralized C was ca. 50%.
4.2. N mineralization
Substantial amounts of N were mineralized during the decomposition of wheat root systems in situ (Fig. 2). But, given the small eects of [CO2] on C
mineral-ization (see above), it is not surprising that we found no eect of [CO2] on N mineralization. This contrasts
with other studies in which signi®cant eects of [CO2]
have been found on N cycling processes such as miner-alization (Hungate et al., 1997b), immobilization
(Tor-bert et al., 1995; Hungate et al., 1997b) and
denitri®cation (Smart et al., 1997; Arnone and Bohlen, 1998; Robinson and Conroy, 1999).
An obvious dierence between those studies and ours is that the latter did not involve living plants. Liv-ing plants aect soil microbial processes by several mechanisms. C losses from living roots can increase the amounts of substrates available to support hetero-trophic microbes (e.g. Smart et al., 1997). Dierential water use by plants grown under elevated [CO2]
in¯u-ences soil moisture and aeration, a particularly import-ant in¯uence on the balance between aerobic and anaerobic microbial processes (e.g. Hungate et al., 1997a; Arnone and Bohlen, 1998; Nicklaus et al., 1998; Robinson and Conroy, 1999). Living roots can also produce speci®c signalling molecules which in¯u-ence microbial metabolism (e.g. Pierson and Pierson, 1996), although the eects of [CO2] on such processes
remain unknown.
[CO2] does not always in¯uence soil microbial
pro-cesses even when living vegetation is present. For example Hungate et al. (1997b) and Nicklaus (1998) found that many N cycle processes in grasslands were aected by [CO2] only if other nutrients were also
available to plants and microbes. Franck et al. (1997) suggested that a large element of species speci®city on the eect of [CO2] on soil N processes is to be
expected. We suggest that such speci®city will apply es-pecially when those processes are measured under liv-ing vegetation, but perhaps less so when livliv-ing plants are absent, as they are in many decomposition studies. Then, the legacy of elevated [CO2] eects on soil N
processes may always be small, irrespective of the taxonomic provenance of the organic matter supplying that N.
We found no eects of [CO2] on N mineralized from
wheat roots decomposing in the absence of living plants. Likewise, Randlett et al. (1996) found none when Populus leaves decomposed in the absence of
4.3. Using13C to determine in situ mineralization of root C
We measured root C mineralization in situ in soil using 13C labelling of roots. The principle of this method is based on the dierence in 13C/12C ratio (expressed in d13C values) between recently grown roots and native SOM. Similar methods have been used in studies of C dynamics in soils with a veg-etation of C3 plants d13C1ÿ32to ÿ20- being replaced by one of C4plants d13C1ÿ17to ÿ9-), or vice versa, with measurements of shifts in d13C of SOM or soil CO2 (see Balesdent and Mariotti, 1996;
HoÈgberg and Ekblad, 1996).
In previous experiments on eects of elevated atmos-pheric [CO2], 13C labelling was used to quantify
pre-sumed [CO2]-induced increases in soil C accumulation
(Leavitt et al., 1994; Hungate et al., 1996; Leavitt et al., 1996). In these free air CO2 enrichment
exper-iments, only CO2 in the elevated [CO2] treatment was
labelled with13C. Ineson et al. (1996) and Nitschelm et al. (1997) grew C3plants on soils containing C derived
substantially from C4 vegetation to enable
measure-ments of soil C input at ambient [CO2] (and ambient
d13C) as well. In our experiments, CO2 at both ambi-ent and elevated [CO2] had a d13C more negative than
that of ambient air. The 13C labelling was not strong enough to allow changes in d13C of total soil organic C to be detected, but it greatly aected d13C of CO2 evolved from soil.
The potential accuracy of the 13C method was demonstrated by the experiment in which C mineraliz-ation was measured on soil only and on soil with known additions of roots (Series (iii)). We measured all the d13C values required to calculate C mineralized from roots (i.e., d13C of added roots, of CO2 evolved from soil plus roots and of CO2 evolved from soil
only). Larger errors may be expected whend13C of the root C source is less well known, as in our incubation of soil after removal of visible roots (Series (ii)). We are unsure why C mineralized from SOM per unit of soil was much smaller in that incubation compared with the incubation of soil with root systems in situ (Table 2). One reason is that the d13C values of extracted roots may have been dierent from those of the actual root-derived microbial substrates, so in¯uen-cing the calculations of C mineralization.
We did not take into account possible 13C/12C frac-tionation during root decomposition, i.e., we used a constantd13C value for the root C source in our calcu-lations of root C mineralized. Two observations suggest that 13C/12C fractionation was indeed negli-gible. First, the demonstrated accuracy of the method as it was used, as discussed above. Second,d13C values of retrieved roots at the end of the decomposition ex-periment were statistically indistinguishable from those
at the start (see Section 3.1). HoÈgberg and Ekblad (1996) measured no 13C/12C fractionation during sucrose decomposition in soil; it seems that the same applies to the decomposition of more complex sub-strates.
5. Conclusions
1. C mineralization from root systems in situ was tran-siently faster after growth at elevated [CO2]
com-pared with ambient [CO2]. Ultimately, however,
there were no [CO2]-related dierences in the
amounts of C or N mineralized from root systems in situ.
2. Rates of C loss from extracted roots per unit root dry weight were not signi®cantly dierent for roots from the two [CO2] treatments.
3. Root decomposition of wheat crops will not necess-arily be aected if grown under elevated [CO2]. Any
[CO2] eects are likely to arise from dierences in
the amounts of root material per unit of soil rather than on the chemical quality of that material. 4. 13C/12C fractionation during root decomposition
was negligible. The `near natural abundance' d13C method can be used to trace root-derived C in soil.
Acknowledgements
Margret Van Vuuren was funded by the Biotechnol-ogy and Biological Sciences Research Council's Bio-logical Adaptations to Global Environmental Change Programme. The Scottish Crop Research Institute receives grant-in-aid from the Scottish Executive Rural Aairs Department. We thank Scott Chasalow, Sigrun Holdhus, Winnie Stein, Mark White, Neil Wilson and Ron Wheatley for their help and Peter Kuikman and Bryan Griths for their comments.
References
Akin, D.E., Rigsby, L.L., Gamble, G.R., Morrison III, W.H., Kimball, B.A., Pinter Jr., P.J., Wall, G.W., Garcia, R.L., LaMorte, R.L., 1995. Biodegradation of plant cell walls, wall carbohydrates and wall aromatics in wheat grown in ambient or enriched CO2concentrations. Journal of the Science of Food and Agriculture 67, 399±406.
Arnone, J.A., Bohlen, P.J., 1998. Stimulated N2O ¯ux from intact grassland monoliths after two growing seasons under elevated at-mospheric CO2. Oecologia 116, 331±335.
potential to aect ecosystem functions through changes in amount and quality of litter. In: Cadisch, G., Giller, K.E. (Eds.), Driven by Nature. Plant Litter Quality and Decomposition. CAB International, Wallingford, pp. 187±200.
Balesdent, J., Mariotti, A., 1996. Measurement of soil organic matter turnover using 13C natural abundance. In: Boutton, T.W., Yamasaki, S. (Eds.), Mass Spectrometry of Soils. Marcel Dekker, New York, pp. 83±111.
Ball, A.S., 1997. Microbial decomposition at elevated CO2 levels: eect of litter quality. Global Change Biology 3, 379±386. Conroy, J., Hocking, P., 1993. Nitrogen nutrition of C3plants at
el-evated atmospheric CO2 concentrations. Physiologia Plantarum 89, 570±576.
Cotrufo, M.F., Gorissen, A., 1997. Elevated CO2 enhances below-ground C allocation in three perennial grass species at dierent levels of N availability. New Phytologist 137, 421±431.
Cotrufo, M.F., Ineson, P., 1995. Eects of enhanced atmospheric CO2and nutrient supply on the quality and subsequent decompo-sition of ®ne roots ofBetula pendulaRoth. and Picea sitchensis
(Bong.) Carr. Plant and Soil 170, 267±277.
Fitter, A.H., Self, G.K., Wolfenden, J., Van Vuuren, M.M.I., Brown, T.K., Williamson, L., Graves, J.D., Robinson, D., 1996. Root production and mortality under elevated carbon dioxide. Plant and Soil 187, 299±306.
Franck, V.M., Hungate, B.A., Chapin III, F.S., Field, C.B., 1997. Decomposition of litter produced under elevated CO2: depen-dence on plant species and nutrient supply. Biogeochemistry 36, 223±237.
Genstat 5 Committee, 1993. Genstat 5 Release 3 Reference Manual. Clarendon Press, Oxford.
Gordon, D.C., Van Vuuren, M.M.I., Marshall, B., Robinson, D., 1995. Plant growth chambers for the simultaneous control of soil and air temperatures and of atmospheric carbon dioxide concen-tration. Global Change Biology 1, 455±464.
Gorissen, A., Van Ginkel, J.H., Keurentjes, J.J.B., Van Veen, J.A., 1995. Grass root decomposition is retarded when grass has been grown under elevated CO2. Soil Biology & Biochemistry 27, 117± 120.
Griths, B.S., Ritz, K., Ebblewhite, N., Paterson, E., Killham, K., 1998. Ryegrass rhizosphere microbial community structure under elevated carbon dioxide concentrations, with observations on wheat rhizosphere. Soil Biology & Biochemistry 30, 315±321. Heal, O.W., Anderson, J.W., Swift, M.J., 1997. Plant litter quality
and decomposition: an historical overview. In: Cadisch, G., Giller, K.E. (Eds.), Driven by Nature. Plant Litter Quality and Decomposition. CAB International, Wallingford, p. 330. HoÈgberg, P., Ekblad, A., 1996. Substrate-induced respiration
measured in situ in a C3-plant ecosystem using additions of C4-sucrose. Soil Biology & Biochemistry 28, 1131±1138.
Hungate, B.A., Jackson, R.B., Field, C.B., Chapin III, F.S., 1996. Detecting changes in soil carbon in CO2enrichment experiments. Plant and Soil 187, 135±145.
Hungate, B.A., Chapin III, F.S., Zhong, H., Holland, E.A., Field, C.B., 1997a. Stimulation of grassland nitrogen cycling under car-bon dioxide enrichment. Oecologia 109, 149±153.
Hungate, B.A., Lund, C.P., Pearson, H.L., Chapin III, F.S., 1997b. Elevated CO2 and nutrient addition alter soil N cycling and N trace gas ¯uxes with early season wet-up in a California annual grassland. Biogeochemistry 37, 89±109.
Ineson, P., Cotrufo, M.F., Bol, R., Harkness, D.D., Blum, H., 1996. Quanti®cation of soil carbon inputs under elevated CO2: C3 plants in a C4soil. Plant and Soil 187, 345±350.
Jackson, R.B., Sala, O.E., Field, C.B., Mooney, H.A., 1994. CO2 alters water use, carbon gain and yield for the dominant species in a natural grassland. Oecologia 98, 257±262.
Jones, T.H., Thompson, L.J., Lawton, J.L., Bezemer, T.M., Bardgett, R.D., Blackburn, T.M., Bruce, K.D., Cannon, P.F.,
Hall, G.S., Hartley, S.E., Howson, G., Jones, C.G., Kampichler, C., Kandeler, E., Ritchie, D.A., 1998. Impacts of rising atmos-pheric carbon dioxide on model terrestrial ecosystems. Science 280, 441±443.
Jongen, M., Jones, M.B., Hebeisen, T., Blum, H., Hendrey, G., 1995. The eects of elevated CO2 concentrations on the root growth ofLolium perenneandTrifolium repensgrown in a FACE system. Global Change Biology 1, 361±371.
Kemp, P.R., Waldecker, D.G., Owensby, C.E., Reynolds, J.F., Virginia, R.A., 1994. Eects of elevated CO2and nitrogen fertili-zation pretreatments on decomposition of tallgrass prairie leaf lit-ter. Plant and Soil 165, 115±127.
Large, E.C., 1954. Growth stages in cereals. Illustration of the Feekes scale. Plant Pathology 3, 128±129.
Leavitt, S.W., Paul, E.A., Kimball, B.A., Hendrey, G.R., Mauney, J.R., Rauschkolb, R., Rogers, H., Lewin, K.F., Nagy, J., Pinter, P.J., Johnson, H.B., 1994. Carbon isotope dynamics of free-air CO2-enriched cotton and soils. Agricultural and Forest Meteorology 70, 87±101.
Leavitt, S.W., Paul, E.A., Galadima, A., Nakayama, F.S., Danzer, S.R., Johnson, H., Kimball, B.A., 1996. Carbon isotopes and car-bon turnover in cotton and wheat FACE experiments. Plant and Soil 187, 147±155.
Nicklaus, P.A., 1998. Eects of elevated atmospheric CO2 on soil microbiota in calcareous grassland. Global Change Biology 4, 451±458.
Nicklaus, P.A., Spinnler, D., KoÈrner, C., 1998. Soil moisture dynamics of calcareous grassland under elevated CO2. Oecologia 117, 201±208.
Nitschelm, J.J., LuÈscher, A., Hartwig, U.A., Van Kessel, C., 1997. Using stable isotopes to determine soil carbon input dierences under ambient and elevated atmospheric CO2conditions. Global Change Biology 3, 411±416.
Norby, R.J., Cotrufo, M.F., 1998. A question of litter quality. Nature 396, 17±18.
O'Neill, E.G., Norby, R.J., 1996. Litter quality and decomposition rates of foliar litter produced under CO2 enrichment. In: Koch, G.W., Mooney, H.A. (Eds.), Carbon Dioxide and Terrestrial Ecosystems. Academic Press, San Diego, pp. 87±103.
Owensby, C.E., Coyne, P.I., Auen, L.M., 1993. Nitrogen and phos-phorus dynamics of a tallgrass prairie ecosystem exposed to elev-ated carbon dioxide. Plant, Cell and Environment 16, 843±850. Pierson, L.S., Pierson, E.A., 1996. Phenazine antibiotic production
in Pseudomonas aureofaciens: role in rhizosphere ecology and pathogen suppression. FEMS Microbiology Letters 136, 101±108. Randlett, D.L., Zak, D.R., Pregitzer, K.S., Curtis, P.S., 1996. Elevated atmospheric carbon dioxide and leaf litter chemistry: in-¯uences on microbial respiration and net nitrogen mineralization. Soil Science Society of America Journal 60, 1571±1577.
Robinson, C.H., Michelsen, A., Lee, J.A., Whitehead, S.J., Callaghan, T.V., Press, M.C., Jonasson, S., 1997. Elevated atmos-pheric CO2aects decomposition ofFestuca vivipara(L.) Sm. lit-ter and roots in experiments simulating environmental change in two contrasting arctic ecosystems. Global Change Biology 3, 37± 49.
Robinson, D., Conroy, J.P., 1999. A possible plant-induced feedback between elevated atmospheric CO2, denitri®cation and the enhanced greenhouse eect. Soil Biology & Biochemistry 31, 43± 53.
Smart, D.R., Ritchie, K., Stark, J.M., Bugbee, B., 1997. Evidence that elevated CO2levels can indirectly increase rhizosphere deni-tri®er activity. Applied and Environmental Microbiology 63, 4621±4624.
Torbert, H.A., Prior, S.A., Rogers, H.H., 1995. Elevated atmos-pheric carbon dioxide eects on cotton plant residue decompo-sition. Soil Science Society of America Journal 59, 1321±1328. Van Ginkel, J.H., Gorissen, A., 1998. In situ decomposition of grass
roots as aected by elevated atmospheric carbon dioxide. Soil Science Society of America Journal 62, 951±958.
Van Ginkel, J.H., Gorissen, A., Van Veen, J.A., 1996. Longterm
de-composition of grass roots as aected by elevated atmospheric carbon dioxide. Journal of Environmental Quality 25, 1122±1128. Van Vuuren, M.M.I., Robinson, D., Fitter, A.H., Chasalow, S.D.,