Plant, Cell and Environment

(2005)

28

, 241–250

© 2005 Blackwell Publishing Ltd

241

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 20042005

282241250Original Article

13

C discrimination in roots and shootsK. Klumpp

et al.

Correspondence: Hans Schnyder. Fax:

+

49 8161 713243; e-mail:schnyder@wzw.tum.de

C-isotope composition of CO

2

respired by shoots and roots: fractionation during dark respiration?

K. KLUMPP, R. SCHÄUFELE, M. LÖTSCHER, F. A. LATTANZI, W. FENEIS & H. SCHNYDER

Plant Science Department, Technische Universität München, D-85350 Freising-Weihenstephan, Germany

ABSTRACT

The CO

2

respired by leaves is

13

C-enriched relative to leafbiomass and putative respiratory substrates (Ghashghaie

et al

.,

Phytochemistry Reviews

2, 145–161, 2003), but howthis relates to the

13

C content of root, or whole plant respi-ratory CO

2

is unknown. The C isotope composition of res-piratory CO

2

(

dddd

R

) from shoots and roots of sunflower(

Helianthus annuus

L.), alfalfa (

Medicago sativa

L.), andperennial ryegrass (

Lolium perenne

L.) growing in a rangeof conditions was analysed. In all instances plants weregrown in controlled environments with CO

2

of constantconcentration and

dddd

13

C. Respiration of roots and shoots ofindividual plants was measured with an open CO

2

exchangesystem interfaced with a mass spectrometer. RespiratoryCO

2

from shoots was always

13

C-enriched relative to thatof roots. Conversely, shoot biomass was always

13

C-depleted relative to root biomass. The

dddd

-difference betweenshoot and root respiratory CO

2

was variable, and negativelycorrelated with the

dddd

-difference between shoot and rootbiomass (

r

2

====

0.52,

P

====

0.023), suggesting isotope effectsduring biosynthesis.

13

C discrimination in respiration (

R

) ofshoots, roots and whole plants (

e

Shoot

,

e

Root

,

e

Plant

) wasassessed as

e

====

(

dddd

Substrate

----

dddd

R

)/(1

++++

dddd

R

/1000), where root andshoot substrate is defined as imported C, and plant sub-strate is total photosynthate. Estimates were obtained fromC isotope balances of shoots, roots and whole plants ofsunflower and alfalfa using growth and respiration datacollected at intervals of 1 to 2 weeks.

e

plant

and

e

Shoot

differedsignificantly from zero.

e

plant

ranged between

----

0.4 and

----

0.9‰, whereas

e

Shoot

was much greater (

----

0.6 to

----

1.9‰).

e

Root

was not significantly different from zero. The presentresults help to resolve the apparent conflict between leaf-and ecosystem-level

13

C discrimination in respiration.

Key-words

:

Helianthus annuus

L.;

Lolium perenne

L.;

Med-icago sativa

L.; allocation;

13

C discrimination; CO

2

exchange; partitioning; photosynthesis; respiration.

INTRODUCTION

The biomass of C

3

plants is depleted in

13

C relative to atmo-spheric CO

2

. This effect is caused mainly by the slower

diffusion of

13

CO

2

relative to

12

CO

2

through stomata, and astrong preference of Rubisco for the light CO

2

species (Far-quhar, O’Leary & Berry 1982). However, the RuBP car-boxylase reaction discriminates much more than CO

2

diffusion. The overall discrimination during photosynthesisvaries as a function of the relative limitations imposed bydiffusion and photosynthetic capacity (Farquhar,Ehleringer & Hubick 1989). Therefore,

13

C discriminationis a useful indicator of the nature of photosyntheticlimitations.

However, fractionation during post-photosynthetic pro-cesses can also affect

d

13

C values of plant biomass. Thus, ithas been shown that different chemical compounds differin their C isotope composition: for instance, lipids and lig-nin are generally

13

C-depleted, whereas non-structural car-bohydrates, such as starch, are usually

13

C-enriched relativeto whole plant biomass (Park & Epstein 1961; Deines 1980;Ghashghaie

et al

. 2003). Differences in the C isotope com-position of organs may result from differences in theirchemical composition (Hobbie & Werner 2004).

13

C fractionation during allocation and partitioning canalso have an effect on the

d

13

C of respiratory CO

2

(e.g.Jacobson

et al

. 1970). Large differences between the

d

13

C ofbiomass or putative substrates (

d

Substrate

) and respiratoryCO

2

(

d

R

) have been reported (range:

+

10.3 to

-

8.1‰, cf.Ghashghaie

et al

. 2003), whereas absence of any effect hasalso been demonstrated (Lin & Ehleringer 1997). The

d

-difference between biomass or substrate and respiratoryCO

2

was termed apparent fractionation (

e

¢

) by Ghashghaie

et al

. (2003), and is a wide-sense definition of

13

C discrimi-nation in respiration. It includes all isotope effects duringallocation and partitioning which lead to a differencebetween the

d

13

C of photosynthetically fixed CO

2

and thatof respiratory CO

2

. Such effects may result from the non-statistical distribution of

13

C in the glucose molecule (Ross-mann, Butzenlechner & Schmidt 1991), isotope effects dur-ing the pyruvate dehydrogenase reaction, and/or thechemical identity of the substrate for respiration (e.g. lipidsor sugars) (Ghashghaie

et al

. 2003). Tcherkez

et al

. (2003)observed a strong relationship between the

d

of CO

2

respired by leaves of

Phaseolus vulgaris

and the respiratoryquotient (RQ; CO

2

produced per O

2

consumed), which wasin accordance with switches between substrates of differentchemical identity and

d

13

C. Similar observations were madeby Jacobson

et al

. (1970) in studies of cyanide effects onrespiration of potato tuber slices.

242

K. Klumpp

et al

.

© 2005 Blackwell Publishing Ltd,

Plant, Cell and Environment,

28,

241–250

Knowledge of

13

C discrimination in respiration is impor-tant, since it could modify the interpretation of biomass

d

13

C in terms of the nature of photosynthetic limitations.Further, if there is a systematic difference between the

d

13

Cof CO

2

fixed in photosynthesis and that of respiratory CO

2

,it could be used to partition the photosynthetic and respi-ratory component of net CO

2

exchange (Yakir & Wang1996; Bowling, Tans & Monson 2001). The finding of asubstantial and systematic

13

C-enrichment in respiratoryCO

2

relative to biomass and different putative substrates inleaves of several herbaceous C

3

dicots reported by Ghash-ghaie and co-workers (Duranceau

et al

. 1999; Duranceau,Ghashghaie & Brugnoli 2001; Ghashghaie

et al

. 2001) istherefore of great interest. Yet, it is currently unknown ifsimilar or divergent effects occur in roots, or at the wholeplant level. Such information is essential for the interpreta-tion of natural abundance of

13

C signatures of respiratoryCO

2

emitted by plants and ecosystems.One great difficulty for the proof of ‘true’

13

C discrimina-tion in respiration is the natural and wide-spread occur-rence of isotopic disequilibria in plants. These are causedby fluctuations in the isotopic composition of photosyn-thate, which arise from changes in photosynthetic

13

C dis-crimination (

D

P

; Farquhar

et al

. 1989), or alterations of the

d

of source CO

2

(

d

C

O

2

). Changes in the

d

13

C of photosyn-thate (

d

P

) are translated into a

d

-change of respiratory CO

2

only with a delay, because of the time needed for the newassimilate to arrive in metabolic pools (e.g. Ekblad & Hög-berg 2001, Pataki et al. 2003; Schnyder et al. 2003). There-fore, fluctuations of dP are not in phase with dR, causing theisotopic non-equilibrium. True 13C discrimination in respi-ration can only be verified and quantified if such isotopicdisequilibria are suppressed in the experiment.

The aims of this study were: (1) to examine whether true13C discrimination occurs during respiration of plants grow-ing in controlled environment with a constant concentra-tion and d13C of CO2; (2) to analyse the relationshipbetween the d13C of shoot and root respiratory CO2; and(3) to explore the consequences of respiratory 13C discrim-ination for the interpretation of overall 13C discriminationat the whole plant level. To this end we took advantage ofa series of experiments (n = 4) that had been conductedwith different objectives, but which all incorporated mea-surements of the d13C of root and shoot respiration. Theseincluded studies with perennial ryegrass (Lolium perenneL.), sunflower (Helianthus annuus L.), and alfalfa (Medi-cago sativa L.) grown in diverse, but constant environmen-tal and C isotopic conditions. Thus, the generality (orvariability) of the above relationships [(1)–(3)] could beassessed.

MATERIALS AND METHODS

Plant material and growth conditions

The study reports on data from four independent experi-ments. These experiments were conducted with differentspecific objectives (see below), but all investigated features

of the C economy of plants, including respiration of wholeshoots (including stems and leaves) and roots.

In all experiments the plants were grown in constantconditions in controlled environment chambers (E15; Con-viron, Winnipeg, Canada) which formed part of a meso-cosm 13CO2/12CO2 gas exchange system described bySchnyder et al. (2003). The experiments included sunflowercv. Optisol, alfalfa cv. Planet, and perennial ryegrass cv.Racolta. All plants were grown in stands, formed by a usu-ally dense (but see below) arrangement of plastic pots(made of polyvinylchloride) holding single plants. The pots(5 cm diameter, 35 cm depth) were filled with washedquartz sand. The plants received a modified Hoagland-typenutrient solution with nitrate as the sole nitrogen sourceseveral times per day (for modifications of the nutrientsolution, see below). The pots were held in plastic contain-ers (76 cm long, 56 cm wide, 32 cm high). Two containerswere placed in each growth chamber.

In all experiments the air supplied to the growth cham-bers was generated from CO2-free air, which was mixedwith CO2 of known d13C using mass flow controllers (MFC).The rate of air supply to each chamber was 250–400 stan-dard litres per minute. Thus, air flow into the chamber wasequal to five to eight times the chamber volume per hour.Chambers received CO2 from one of two CO2 sources,either of mineral (relatively 13C-rich) or fossil-organic ori-gin (relatively 13C-depleted; all from Messer Griesheim,Frankfurt, Germany). In all experiments the d13C and con-centration of CO2 inside the chambers was kept near con-stant by periodic adjustments of airflow and of the CO2

concentration at the chamber air inlet. Experiments I, IIand III are described in Lötscher, Klumpp & Schnyder(2004). Details of experiment IV are given in Lattanzi,Schnyder & Thornton (2004).

Experiment I (sunflower)

This study investigated effects of plant–plant competitionfor light on C allocation, partitioning and respiration ofsunflower plants. To this end stands of sunflower wereestablished and grown at densities of 400 (high density) and50 plants m-2 (low density). The high density stand was a50 : 50 mix of two sub-populations: plants sown early andplants sown with a delay of 1 week. The former grew tallerand, hence, dominant as a result of the head start, whereasthe latter became subordinate. Only results from theformer are reported here, as the subordinate plants exhib-ited very small respiration. Throughout the experiment adrip irrigation system supplied a half-strength Hoagland-type solution with 5.0 mmol L-1 nitrate-N to every potevery 4 h. Light was supplied by cool white fluorescenttubes and incandescent lamps, providing a 16 h light periodwith a photosynthetic photon flux density (PPFD) of700 mmol m-2 s-1 at the top of the canopy. The temperaturewas set at 20/16 ∞C and relative humidity at a constant 70%during the light and dark periods. The concentration andd13C of CO2 in the chambers was kept constant near350 mL L-1 and -1.8‰. Respiration was measured and

13C discrimination in roots and shoots 243

© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 241–250

plants sampled at weekly intervals, between 3 and 5 weeksfrom germination.

Experiment II (alfalfa)

This experiment was performed to assess effects of compe-tition on C allocation, partitioning and respiration. This wasdone with singly potted individuals of alfalfa growing indense pure stands (400 plants m-2). Stands contained a50 : 50 mixture of plants originating from two pretreat-ments: plants established at a moderate irradiance(350 mmol m-2 s-1 PPFD at pot height during a 16 h day for4 weeks from sowing) and cut to a stubble height of 8 cm,and plants established at low irradiance (100 mmol m-2 s-

1 PPFD) and cut to a stubble height of 3 cm, at the time ofassembling the stands. The containers holding stands wereflooded twice a day for 1 h with a half-strength modifiedHoagland solution containing 7.5 mmol L-1 nitrate-N. Oncea week the pots were flushed with water. Growth chamberswere adjusted to 22/18 ∞C day/night temperatures, 75% rel-ative humidity, and 350–500 mmol m-2 s-1 PPFD at canopyheight during the 16 h photoperiod. It was expected thatthe pretreatments would result in plants of different height.However, all plants reached the top of the canopy. The CO2

concentration in all chambers was held constant near350 mL L-1. Two chambers were supplied with CO2 havinga d13C of -2.0‰, whereas the other two received CO2 witha d13C near -43.7‰. Respiration was measured and plantssampled at weekly intervals starting at 2 until 4 weeks afterassembling the stands.

Experiment III (alfalfa)

This study was conducted to simulate effects of light and Ncompetition on C allocation, partitioning and respiration.To this end 3-week-old plants of alfalfa were cut to a 3-cmstubble height and assigned and exposed to one of fourcombinations of high or low irradiance (I+/I–: 610 or90 mmol m-2 s-1 PFD at canopy height), and high or lownitrogen supply (N+/N–: 7.5 or 1.5 mM L-1 of nitrate-N inthe nutrient solution) for 5 weeks. In all treatments theconcentration and d of CO2 in the chambers was kept con-stant near 350 mL L-1 and -2.4‰. Respiration was mea-sured and plants sampled at 5 weeks after imposition oftreatments.

Experiment IV (ryegrass)

In this study we analysed effects of light competition ongrowth and recovery from defoliation in ryegrass andPaspalum dilatatum Poir. Briefly, a 50 : 50 mixed stand withplants of the two species was grown at 15/14 ∞C day/night,a vapour pressure deficit of 0.5/0.3 kPa, and 550 mmol m-2

s-1 PPFD at the top of the canopy during the 12 h photope-riod. All plants grew singly in pots. Stands were floodedfour times a day for 30 min with a half-strength modifiedHoagland solution containing 7.5 mmol L-1 nitrate-N.Industrial CO2 with d13C -47.0‰ was used as a CO2 source.

The concentration and d13C of CO2 inside the chamber waskept near 300 mL L-1 and -47.0‰ by periodic adjustmentsof airflow and of the CO2 concentration in the inlet air.Respiration measurements were performed and plantssampled 9 week after sowing. Paspalum plants were toosmall and respired very little. Thus, only the ryegrass respi-ration data are presented.

Respiration measurements and isotope analysis

Respiratory 13CO2/12CO2 exchange system

A detailed description of the respiration measurement sys-tem was recently given by Lötscher et al. (2004). Briefly,root and shoot respiration as well as the d13C of root andshoot respiratory CO2 of individual plants was measured inan open gas exchange system. The system included fourcuvettes, each of which consisted of a tube and a top andbottom plate (all made of polyvinylchloride) (Fig. 1), whichcould be opened and closed quickly to insert a pot. A bush-ing that matched exactly the size of the pot was glued intothe bottom plate. A similar system was used to seal thebottom of the pot. Rubber seals and vacuum grease wereused to ascertain that the cuvettes were gas tight. The sys-tem was connected to an infrared gas analyser (IRGA) anda continuous-flow stable isotope-ratio mass spectrometer(CF-IRMS) via Teflon tubes. Air with known constant d13C

Figure 1. Scheme of respiration cuvette. For further details see Materials and methods section.

244 K. Klumpp et al.

© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 241–250

and concentration of CO2 (200 mL L-1) was supplied tocuvettes at a rate of 0.3–1.0 L min-1 after passage of ahumidifier. Air flow was controlled by MFCs. Cuvetteswere arranged in a plant growth cabinet held at the sametemperature as plant growth chambers. Each cuvette hadtwo outlets: one in the shoot section on the opposite endof the inlet, and the other at the bottom of the pot whichenclosed the root compartment. Air in the shoot compart-ment was ventilated by a fan. About 0.15–0.45 L min-1 ofthe cuvette air was drawn through the root compartmentwith a gas-tight Teflon-lined peristaltic membrane pump.The air was then dried and the flow to a multiway valveblock (sample air selector, SAS) controlled by a MFC. Theremaining air from the shoot compartment was directlyconveyed to the SAS. A reference air line (0.9 L min-1) wasalso connected to the SAS. The SAS sequentially sampledthe reference air line and the different sample air lines andfed the air to the IRGA and IRMS as described in Schnyderet al. (2003). A full measurement cycle of all four cuvetteswas completed in about 30 min. Readings of the IRGA andIRMS were continuously stored in files. The standard devi-ation associated with the measurement of d13C and CO2

concentration of the reference air CO2 was typically<0.15‰ and <1 mL L-1.

Protocol

Plants were removed from the growth chambers at the endof the light period, pots flushed with 0.5 L water and thenrinsed with nutrient solution, which was previously aeratedwith CO2-free air for 3 d. Thereafter, the pots wereenclosed in the respiration cuvettes. Air injection in theshoot compartment was initially set at 3 L min-1. When theCO2 concentration in the exit of the shoot compartmenthad decreased to a near-constant concentration, the drain-age of the root compartment was opened and the air fittinginstalled. Air was then drawn through the root compart-ment at 1 L min-1 until the CO2 concentration at the rootexit became near constant. These procedures aimed atremoving all extraneous CO2 from the shoot and root com-partment as quickly as possible, and thus minimize the timelag between insertion of a pot and collection of first reliabledR data. System tests demonstrated that up to 1 h wasrequired to purge the system from all extraneous CO2

(Fig. 2). Tests also revealed that the system did not fraction-ate 13C: when pots with clean wet sand were inserted in thesystem, the d13C of CO2 at the root outlet (following purg-ing of the system) did not differ from that at the cuvetteinlet (Fig. 2). When CO2 concentration readings hadbecome near stable the flow rates were set correspondingto the plant size. First measurements of shoot and rootrespiration and d were obtained at about 1 h after the plantswere removed from the stands. Respiration was measuredthroughout the regular duration of the dark period. The Cisotope composition of CO2 respired by shoots and rootswas relatively constant during a dark period (Fig. 3). Thatis, any trends over time of the d of CO2 respired by shootsand roots were statistically non-significant. Flux-weighted

mean d13C of respiratory CO2 collected throughout the darkperiod are presented in this paper.

Plant sampling, and elemental and isotope analysis

Plants were separated into shoot [including all leaf andstem tissue (reproductive organs had not formed)] and rootfractions immediately after the termination of respirationmeasurements. The two fractions were dried (30–60 min at100 ∞C, and then at 65 ∞C for 48 h) in a forced draught oven,weighed, and ground to a fine powder in a ball mill. The Ccontent and d13C of 1 mg aliquots of samples was deter-mined by a elemental analyzer (Carlo Erba NA1110, Mil-ano, Italy) interfaced to the IRMS.

All isotope data were expressed in the conventional d13C-notation (in ‰) relative to a VPDB-gauged standard as

Figure 2. Time course of the concentration (a) and d13C (b) of CO2 entering the respiration cuvette (�), and leaving it at the root outlet (�,�) of two pots. Measurements were done with pots con-taining clean sand. Time refers to the closure of the system when the top plate (see Fig. 1) was fixed following insertion of the pots. Pots were flushed either with CO2-free nutrient solution (�), or normal nutrient solution (�) before inserting them into the respi-ration chamber. The d13C of CO2 at the chamber inlet and outlets did not differ after 1 h: � -49.03‰ (± 0.13), � -48.95‰ (± 0.10), � -48.83‰ (± 0.09).

13C discrimination in roots and shoots 245

© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 241–250

dsample = 1000(RSample/Rstandard - 1) (McKinney et al. 1950),where R is the 13C/12C ratio in the sample or the standard.

Data analysis: gas exchange parameters, growth analysis, allocation model, 13C fractionation

Respiration of roots (RRoot) and shoots (RShoot) and the d13Cof the respiratory CO2 evolved from roots (dR Root) andshoots (dR Shoot) were obtained as

Rshoot = Fin(Cin - CS out) (1a)

Rroot = FR out(CS out - CR out) (1b)

dR Shoot = [(dS out Fin CS out) - (din Fin Cin)]/RShoot (2a)

dR Root = [(dR out FR out CR out) - (dS out FR out CS out)]/RRoot (2b)

with F the flux of air at standard humidity (mol s-1), C theconcentration of CO2 in air (mol mol-1), and subscripts ‘in’,‘R out’ and ‘S out’ designating the respiration chamberinlet, root compartment outlet and shoot compartment out-let, respectively.

The C isotope composition of C accumulating in roots(dRoot) and shoots (dShoot), of C imported in roots (dI Root) andshoots (dI Shoot), as well as of the total photosynthate (dP)produced by individual plants during a given interval i - 1to i was estimated using the data from the periodic samplingand C elemental and isotope analysis of roots and shoots,and of the concurrent respiration measurements in experi-ments I and II. Thus, for dRoot and dShoot

dShoot = (dShoot ti Cshoot ti - dShoot ti - 1 Cshoot ti - 1)/(Cshoot ti - Cshoot ti - 1) and (3a)

dRoot = (dRoot ti Croot ti - dRoot ti - 1 Croot ti - 1)/(Croot ti - Croot ti - 1)(3b)

with dShoot ti, Cshoot ti, dRoot ti, and Croot ti representing the Cisotope composition and C mass of shoots and roots atsampling date i, and dShoot ti-1, Cshoot ti-1, dRoot ti-1, and Croot ti-1

the same parameters for the previous sampling date.The d of C imported in roots and shoots was calculated

as the weighted average d13C of C accumulated and respiredin root and shoots

dI Shoot = [dShoot (dCShoot/dt) + dR Shoot RShoot)]/IShoot and (4a)

dI Root = [dRoot (dCRoot/dt) + dR Root RRoot)]/IRoot (4b)

with IShoot and IRoot, the rate of C import in shoots and roots,as obtained from the rates of C accumulation and respira-tion: Ishoot = dCShoot/dt + RShoot, and Iroot = dCRoot/dt + RShoot,respectively. dCShoot/dt was calculated as dCShoot/dt = (Cshoot ti - Cshoot ti-1)/[ti - (ti - 1)]. dCRoot/dt was obtainedsimilarly.

The estimate of R included respiration during darknessand daylight hours, where the latter was estimated fromrespiration in darkness and temperature, assuming aQ10 = 2, for short-term, that is, diurnal fluctuations of R.This was verified for sunflower and alfalfa, by continuousmeasurements of root respiration (data not shown). It wasfurther assumed (a) that the d13C of respiratory CO2 wasthe same in light and darkness, and (b) that losses of C inthe form of exudates and volatile organic C were negligible.Dead tissue (mainly leaves) was included in the sampling.

The d13C of total photosynthate (dP) produced by theplant during the interval i - 1 to i, was obtained as

dP = (dI Shoot Ishoot + dI Root IRoot)/Pn, (5)

with Pn = Ishoot + IRoot.Apparent fractionation (e, ‰) during respiration in the

shoot (eShoot) and root (eRoot) was estimated according to therelationship e = (dSubstrate - dProduct)/(1 + dProduct/1000), wherethe d13C of the substrate in the root or shoot was equatedwith that of total C import. Thus

eShoot = (dI Shoot - dR Shoot)/(1 + dR Shoot/1000) and (6a)

eRoot = (dI Root - dR Root)/(1 + dR Root/1000) (6b)

Accordingly, apparent fractionation during respiration atthe whole plant level (ePlant) was obtained as

ePlant = (dP - dR Plant)/(1 + dR Plant/1000) (7)

with dR Plant the respiration-weighted average d13C of rootand shoot respiration.

Statistical analysis

Analyses of variance and t-test of paired samples of d13Cbulk and respired C of shoot and root, respectively, wereperformed using procedure ANOVA and t-test for pairedsamples of STATISTICA (Statistica 5; Statsoft, Inc., Tulsa,OK, USA).

Figure 3. Time course of the d13C of CO2 respired by shoots (�, �) and roots (�, �) of sunflower during the regular dark period of the day. Plants were grown at low (�, �) and high (�, �) density (experiment I). Means ±SE.

246 K. Klumpp et al.

© 2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 241–250

RESULTS

Relationships between dddd13C of shoot and root respiratory CO2 and dddd13C of biomass

Shoot respiratory CO2 was always 13C-enriched relative toshoot biomass (+2.6‰ ± 0.4) (Table 1). In contrast, root res-piratory CO2 was always 13C-depleted relative to root bio-mass (-2.7‰ ± 0.4). Because of the opposing relationshipsin shoots and roots, the discrepancy was much smaller atthe whole plant level: respiratory CO2 evolved by the wholeplant was on average 1.1‰ enriched in 13C relative to totalplant C.

The relationship between the d13C value of respiratoryCO2 and biomass varied among species and treatments(Table 1). Since the different species were grown in differ-ent conditions (light, N nutrition, temperature), it is unclearwhether species’ differences had a genetic component.However, the large variation in the relationship betweenthe d13C of biomass and respiratory CO2 in the differenttreatments of alfalfa suggested a strong influence of growthconditions.

There was also a striking difference between the d13Cvalues of CO2 respired by shoots and roots, with shootrespiratory CO2 always 13C-enriched relative to root respi-ratory CO2 (Table 1). On average of species and treatmentsthe d13C of shoot respiratory CO2 was 4.1‰ enriched in 13Crelative to that of roots. Again, there was substantial vari-ation in this relationship (2.4 to 7.6‰). The d-differencebetween shoot and root respiratory CO2 was largest inryegrass, and smallest in sunflower, but almost the entirerange of variation was found among the different treat-ments of alfalfa. The d13C values of shoot and root biomassalso differed, and this difference was opposite to that of therespiratory CO2 (Table 1). That is, the shoot biomass wasalways 13C-depleted relative to the root. On average, thisdifference was -1.2‰. Again, the relationship was variable.Importantly, the d-difference between shoot and root bio-

mass was inversely related to the d-difference betweenshoot and root respiratory CO2 (r2 = 0.52, P = 0.023)(Fig. 4).

13C discrimination in respiration of shoots, roots and whole plants

In experiments I (sunflower) and II (alfalfa) respiration wasmeasured and plants sampled at intervals, thus allowing

Table 1. d-differences among respiration and biomass components of sunflower, alfalfa and perennial ryegrass: difference between shoot biomass-d13C and shoot respiratory CO2-d13C (dShoot - dR Shoot), difference between root biomass-d13C and root respiratory CO2-d13C (dRoot - dR Root), difference between shoot and root biomass-d13C (dShoot – dRoot), difference between shoot respiratory CO2-d13C and root respiratory CO2-d13C (dR Shoot - dR Root), and difference between whole plant biomass-d13C and whole plant respiratory CO2-d13C

dShoot - d R Shoot dRoot - dR Root dShoot - dRoot dR Shoot - d R Root dPlant - dR Plant

SunflowerLow density stand -2.70 (0.25) 0.46 (0.37) -0.80 (0.19) 2.38 (0.46) -1.83 (0.16)High density stand -2.02 (0.28) 2.00 (0.30) -0.91 (0.09) 3.11 (0.42) -1.24 (0.21)

AlfalfaLow light pretreatment -2.34 (0.56) 2.95 (0.91) -1.59 (0.34) 3.70 (0.79) 0.03 (0.59) n.s.High light pretreatment -1.90 (1.02) 1.52 (0.29) n.s. -1.00 (0.22) 2.42 (1.20) -0.81 (0.49) n.s.High light/high nitrogen -1.06 (0.20) 3.73 (0.42) -1.03 (0.15) 3.76 (0.18) 0.16 (0.28) n.s.High light/low nitrogen -1.60 (0.41) 2.84 (0.38) -0.95 (0.38) 3.48 (0.10) -0.18 (0.29) n.s.Low light/high nitrogen -5.64 (1.12) 2.39 (1.15) -1.38 (0.10) 6.65 (2.07) -3.94 (0.79)Low light/low nitrogen -2.18 (0.58) 2.68 (0.64) -1.19 (0.06) 3.68 (1.15) -0.76 (0.32) n.s.Perennial ryegrass -3.74 (0.37) 5.39 (0.19) -1.53 (0.53) 7.60 (0.82) -0.97 (0.32)

Means and (SE). Differences are statistically different from 0 at P < 0.05, except where indicated by n.s.

Figure 4. d-difference between CO2 respired by shoots and roots versus d-difference between biomass C of shoots and roots. Results from experiments with sunflower (�), alfalfa (�) and perennial ryegrass (�). Means ±SE.

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construction of C isotope balances for periods of 7–14 d.These data indicated that the d13C of C concurrently accu-mulating in roots and shoots did not differ significantly(Table 2). Moreover, the d13C of C imported into roots andshoots of alfalfa did not differ significantly, although theydid in sunflower. In the latter the d13C of C imported intoroots was approximately 1‰ depleted relative to that in theshoot (P < 0.01). In both experiments the respiratory CO2

evolved from roots was consistently 13C-depleted relativeto that of shoots.

Apparent 13C discrimination in respiration of shootsaveraged -1.3‰, meaning that respiratory CO2 was 13C-enriched relative to imported C. The 13C discrimination inrespiration of roots was insignificant, with one exception:the small (0.6‰) 13C discrimination in root respiration ofalfalfa growing in the presence of CO2 with a d13C -4‰ wasstatistically significant. However, plants growing underidentical conditions but exposed to CO2 with a d13C of -47‰ did not demonstrate 13C discrimination in root respi-ration. Clearly, if 13C discrimination did occur in root res-piration, then it was small.

At the whole plant level 13C discrimination in respirationwas slightly negative (average -0.6‰). Thus, the total res-piratory CO2 produced by plants was slightly 13C-enriched.This effect was significant in two of the data sets (sunflowerat high density, and alfalfa growing in dCO2 -47‰), and overall experiments (-0.7‰, P < 0.01).

DISCUSSION

Contrasting respiratory fractionation in shoots and roots

This study revealed a contrast in apparent respiratory 13Cfractionation in roots and shoots of three herbaceous spe-

cies: a annual and a perennial dicot, and a monocot. Thisphenomenon has not been studied and reported before.CO2 respired by roots was always 13C-depleted relative tothat of shoots in a large range of environmental conditions,indicating that the effect was not the result of singularexperimental conditions, but had a more general validity.

Importantly, the fractionation effects observed in thisstudy were not a result of isotopic disequilibria betweencurrent photosynthate and respiratory substrates. Such dis-equilibria commonly occur in natural situations due to fluc-tuations in the d13C of photosynthate and delays in turnover of the respiratory pools by the products of currentphotosynthesis (Pataki et al. 2003). The d13C of CO2 andenvironmental conditions were constant in each experi-ment, thus minimizing opportunities for the formation ofisotopic disequilibria. Isotopic disequilibria between cur-rent photosynthate and respiratory pools could still arise inconstant conditions, if there were ontogenetic shifts in pho-tosynthetic discrimination. However, we found no indica-tion for such effects in our data: d13C of biomass and ofimported C were constant during the experimental periods.Furthermore, the fractionation effects at the level of thewhole plant and shoot persisted, when isotope balanceswere calculated for discrete periods.

The relative 13C-enrichment of CO2 respired by shoots isin agreement with the findings of Ghashghaie and co-work-ers (Ghashghaie et al. 2003), which demonstrated that CO2

respired by leaves in the dark was 6‰ enriched comparedto leaf sucrose in intact French bean (Phaseolus vulgaris)(Duranceau et al. 1999). Similar results were also obtainedin tobacco (Nicotiana sylvestris) and sunflower (Helianthusannuus) though the respiratory CO2 was slightly lessenriched in 13C (Ghashghaie et al. 2001). Ghashghaie andco-workers also observed variability in 13C-enrichment ofrespiratory CO2, which could be induced in the short term

Table 2. Mean C isotope composition (d13C) of total C accumulated, imported and respired during 1- to 2-week intervals in shoots and roots of sunflower and alfalfa, and respiratory 13C discrimination in shoots, roots and whole plants

Sunflowerhigh density

Sunflowerlow density

AlfalfadCO2 -2‰

Alfalfa dCO2 -43.7‰

d13C of accumulated C Shoot -23.40 (0.48) -24.33 (0.23) -23.33 (0.30) -66.91 (0.31)Root -22.03 (0.84) -24.39 (0.23) -22.40 (0.26) -67.10 (1.93)Diff n.s. n.s. n.s. n.s.

d13C of imported C (dI) Shoot -22.47 (0.19) -23.19 (0.17) -22.94 (0.14) -65.89 (0.13)Root -24.05 (0.36) -23.82 (0.05) -23.12 (0.28) -66.86 (0.79)Diff ** ** n.s. n.s.

d13C of respired C (dR) Shoot -21.71 (0.03) -21.42 (0.08) -22.35 (0.31) -64.05 (0.41)Root -24.94 (0.05) -23.50 (0.07) -23.70 (0.30) -67.36 (0.52)Diff ** ** * *

Fractionation eShoot *-0.77 (0.20) **-1.81 (0.24) -0.61 (0.36) **-1.97 (0.48)eRoot 0.92 (0.35) -0.33 (0.12) *0.60 (0.12) 0.30 (0.30)ePlant -0.42 (0.21) ** -1.35 (0.20) -0.04 (0.20) *-0.68 (0.17)

SE values are given in parentheses. The d13C of C accumulation, respiration and import were calculated by isotopic mass balance as explainedin Materials and Methods. Data from experiments I and II. Data from the two pretreatments in experiment II were merged by dCO2 duringgrowth. **, * and n.s. indicate significance at the P < 0.01, P < 0.05 level and non-significance for the difference between shoot and root d13C,or for e being different from zero.

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and was correlated with changes in the respiratory quotient(RQ). This result suggested switches between different sub-strates for respiration, and was also consistent with the d13Cof putative substrates extracted from the same leaves (Tch-erkez et al. 2003). Our results confirm and extend the find-ing of Ghashghaie in that they show 13C-enrichment ofrespiratory CO2 evolved from shoots in other species, anddemonstrate its occurrence in tightly controlled constantenvironmental and isotopic conditions.

The definition of respiratory fractionation (e) used in thepresent study differs somewhat from that of Ghashghaieand co-workers. They estimated e as the d-differencebetween respiratory CO2 and total (leaf) tissue C or puta-tive substrates (e.g. sucrose), which might already be mod-ified somewhat by the respiratory 13C discrimination. Here,we estimate e from the relationship of respiratory CO2 withtotal photosynthates available for growth, storage andmaintenance in the relevant plant part or whole plant. Wefeel that this definition is appropriate to the scale of thisstudy, particularly since the identity and activity of the dif-ferent respiratory activities in the shoot and root are uncer-tain, and the true chemical identity of the substrates andtheir compartmental distribution is unknown.

The present results suggest a connection between dis-crimination in respiration and biosynthesis. A corollary ofrespiratory 13C fractionation is that it changes the 13C sig-nature of the remaining material in the opposite direction(Ghashghaie et al. 2003). In the present study the contrastin the isotopic signature of shoot and root respiratory CO2

was opposite to the contrast of the C isotope signatures ofshoot and root biomasses: whereas the respiratory CO2 ofshoots was 13C-enriched relative to that of roots, the biom-ass of shoots was 13C-depleted relative to that of roots.Importantly, the difference between shoot and root respi-ratory d13C was negatively correlated with the differencebetween shoot and root biomass d (Fig. 4). This relationshippassed near the origin and had a slope of approx. -3.6 (ifforced through the origin), meaning that for every 1‰change in the relationship between root and shoot biomassd13C, the d-difference between shoot and root respiratoryCO2 changed by 3.6‰. This relationship is consistent withthe view that respiratory discrimination is associated withbiosynthesis, and a growth yield (biomass-C produced perunit C consumed) of approximately 0.78. This was in agree-ment with experimental estimates of the growth yield ofthese plants (0.72 and 0.8 for alfalfa I+/N+, I–/N+ and sun-flower at high density, respectively) by Lötscher et al.(2004) and theoretical estimates (Penning de Vries et al.1974; Johnson 1990; Amthor 2000; Cannel & Thornley2000). The absence of discrimination in respirationobserved in studies with protoplasts by Lin & Ehleringer(1997) could be linked to an absence of biosynthetic activityof the protoplasts.

Support for a causal relationship between 13C discrimina-tion in respiration and biosynthetic processes is alsoobtained from the analysis of other data. Thus, others havenoted a similar difference between root and shoot biomassd13C (Bradford, Sharkey & Farquhar 1983; Yoneyama &

Ohtani 1983; Viktor & Cramer 2003; Hobbie & Werner2004). Calculations using the chemical composition of rootsand shoots as reported by Poorter & Bergkotte (1992)together with the (average) deviation of C isotope signa-tures of chemical compound classes relative to bulk C ascompiled by Gleixner et al. (1993) and Schmidt & Gleixner(1998) give a d-difference of 1 and 2‰ between the shootand root for fast- and slow-growing herbaceous species.

However, other mechanisms than respiratory fraction-ation in biosynthetic processes could also contribute to thedifference in respiratory 13C fractionation between shootsand roots. For instance, re-assimilation of respiratory CO2

in roots by PEPc could add to the contrast of d13C in respi-ratory CO2 released by roots and shoots, because the sub-strate for PEPc (HCO3

–) is 13C-enriched relative to the CO2

pool from which it is formed. Thus, the respiratory CO2

escaping from the roots would be 13C-depleted relative tothe respiratory substrate, increasing the d-differencebetween respiratory CO2 evolved from shoots and roots.This would also increase the difference in d13C betweenbiomass C and respiratory CO2 of roots. Moreover, if the13C-enriched organic acids were transported to and decar-boxylated in the shoot, the contrast between shoot and rootrespiratory CO2 d13C would be further enhanced. Supportfor such a mechanism comes from the observation of astimulated translocation of organic acids from roots toshoots by nitrate nutrition (Cramer & Lips 1995), and a RQof approximately 1.2 for dark respiration of leaves in asignificant fraction of the data reported by Tcherkez et al.(2003). However, more detailed studies of PEPc functionin roots and shoots are needed to verify a role of PEPc indetermining the d13C of respiratory CO2 released fromshoots and roots.

Allocation also had some effect on the d13C of respiratoryCO2 of roots and shoots: on average of all treatments, Cimported into the root was 13C-depleted by 0.8‰ relativeto the C imported into the shoot (P < 0.05; Table 2). Thiseffect could result from different allocation patterns of Cfixed by well-lit and shaded leaves, and a differencebetween the two leaf categories in the d13C of photosyn-thate produced by them. In a 14C-labelling study withtomato Shishido, Kumakura & Nishizawa (1999) observedthat lower leaves allocated relatively more C to roots thanupper leaves; whereas upper leaves allocated relativelymore C to the shoot apex. Photosynthetic 13C-discrimina-tion in sun leaves is less than in shaded leaves, leading tomore negative d13C of assimilate produced by shaded thanby well-lit leaves (LeRoux et al. 2001; Baldocchi & Bowling2003).

Plant level respiratory 13C discrimination

The results demonstrated that, if the proportions of theCO2 respired by different plant parts and proportions ofcarbon allocated to these different parts were consideredin a mass balance, there was a small plant-level respiratory13C discrimination. Plant-level respiratory 13C discrimina-tion was small, because of the opposing behaviour of shoots

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and roots. Still the effect was significant (P < 0.01) and aver-aged -0.7‰ meaning that respiratory CO2 was somewhat13C-enriched relative to photosynthetic products. This effectwas nearly identical with that of perennial ryegrass standsdetermined by other means, i.e. by continuous 13CO2/12CO2

gas exchange measurements of whole stands in light anddarkness (Schnyder et al. 2003). Since respiration con-sumed about 45% (± 0.04) of total photosynthate in thepresent experiments, the effect of respiratory 13C discrimi-nation on the d13C of total biomass was small: on averageof the different experiments it would have shifted the d13Cof biomass by less than 0.3‰ relative to the primary pho-tosynthetic products. One implication of this finding is thatneglecting respiratory 13C discrimination leads to only smallerrors in the interpretation of biomass-d13C in terms of thecontribution of stomatal and photosynthetic capacity limi-tations on photosynthesis, if it is based on whole plant d13C.Because of its small magnitude the usefulness of ePlant forpartitioning stand-scale respiratory and photosyntheticCO2 fluxes during isotopic equilibrium periods seemsgreatly limited. So, partitioning based on natural abun-dance isotope signatures would need to rely on isotopicdisequilibria which naturally arise from changing environ-mental conditions, but which may vary greatly in magni-tude. Yet, such environmental perturbations might entrainparallel changes in allocation, and shifts in the relative mag-nitudes of root and shoot respiration. Such effects couldcontribute to changes in the d13C of plant level respirationvia their (shoot and root) specific e. But whether or not sucheffects really occur remains to be tested.

In conclusion, the finding of Ghashghaie and co-workersof a substantial (negative) 13C discrimination in respirationof leaves is validated by this study at the whole shoot level.However, the effect was strongly counterbalanced by rootswhich tended to produce 13C-depleted respiratory CO2,thus yielding only a small respiratory 13C discrimination atthe whole plant level. The present results help to resolvethe apparent conflict between leaf- and ecosystem-level 13Cdiscrimination in respiration.

ACKNOWLEDGMENTS

This work was supported by the Deutsche Forschungsge-meinschaft (SFB 607). Jaleh Ghashghaie is thanked forvaluable comments on an earlier draft of the paper. We alsothank two anonymous reviewers for thoughtful comments.

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Received 24 May 2004; received in revised form 23 September 2004;accepted for publication 27 September 2004