Plant material and growth conditions
Seeds of perennial ryegrass (L. perenne L., cv. Acento) were sown individually in plastic pots filled with washed quartz sand and arranged in plastic containers at a density of 378 plants m−2. Two containers were placed in each of four growth chambers (Conviron E15, Conviron, Winnipeg, Canada). Plants were grown in continuous light, supplied by cool white fluorescent tubes. Irradiance was maintained at 275 µmol m−2 s−1 photosynthetic photon flux density at plant height. Temperature was controlled at 20 °C and relative humidity near 85%. Water and nutrients were supplied by briefly flooding the containers every 3 h.
Half the stands received a modified Hoagland solution containing 1 mm NO3- (low-nitrogen plants), with 1 mm KNO3, 1 mm MgSO4, 0.18 mm KH2PO4, 0.21 mm K2HPO4, 0.5 mm NaCl, 0.7 mm K2SO4 and 2 mm CaCl2; micronutrients: 125 µm Fe-ethylenediaminetetraacetic acid, 46 µm H3BO3, 9 µm MnSO4, 1 µm ZnSO4, 0.3 µm CuSO4 and 0.1 µm Na2MoO4. The other stands received a nutrient solution containing 7.5 mm NO3- (high-nitrogen plants) with 2.5 mm Ca(NO3)2, 2.5 mm KNO3, 1.0 mm MgSO4, 0.18 mm KH2PO4, 0.21 mm K2HPO4, 0.5 mm NaCl, 0.4 mm KCl, 0.4 mm CaCl2, and the same levels of micronutrients. All stands were periodically flushed with demineralized water to prevent salt accumulation.
CO2 control in the growth chambers and 13C labelling
The labelling system described by Schnyder et al. (2003) with air-locks as detailed by Lehmeier et al. (2008), was used. In brief, air supply to the growth chambers was performed by mixing CO2-free air and CO2 of known carbon isotope composition (δ, with δ = [13C/12Csample/13C/12CVPDB standard] − 1). Both δ13C and concentration of CO2 (360 µL L−1) inside the chambers were constantly monitored by an infrared gas analyser (Li-6262, Li-Cor Inc., Lincoln, NE, USA) and a continuous-flow isotope-ratio mass spectrometer (CF-IRMS, Delta Plus, Finnigan MAT, Bremen, Germany). At high nitrogen supply, half the stands grew with 13C-depleted CO2 (δ13C −28.8‰ ± 0.2 SD), while the other half grew with 13C-enriched CO2 (δ13C −1.7‰ ± 0.2). For stands of low nitrogen supply, δ13C was −3.6‰ (±0.2) and −30.9‰ (±0.3), respectively (CO2 from Linde AG, Höllriegelskreuth, Germany). The stability of the isotopic composition and concentration of CO2 (±3 µL L−1 SD on average over all measurements in one chamber) inside the chambers was provided by periodic adjustments of airflows and CO2 concentrations in chamber inlets.
When plants had three tillers (about 3 and 6 weeks after sowing at high and low nitrogen supply, respectively), labelling was initiated by swapping randomly selected individual plants between chambers of the same nitrogen supply level (i.e. 13C-enriched CO213C-depleted CO2 and vice versa). This ensured that high- and low-nitrogen plants were compared at a similar size, so that possible size-related effects on the respiratory supply system were minimized. Plants at high nitrogen supply were kept in the labelling chamber for 1, 2, 4, 8 or 16 h; or for 1, 2, 4, 8, 12, 17 or 25 d. The durations of labelling at low nitrogen supply were 1, 2, 4, 8 or 16 h; or 1, 2, 4, 8 or 29 d. Within one nitrogen supply level, labelling was scheduled in such a way that respiration measurements occurred at the same mean plant age (and size).
Respiration of labelled and non-labelled (control) plants was measured in the system described by Lötscher, Klumpp & Schnyder (2004) and Klumpp et al. (2005). Briefly, four plants were removed from the stands, rapidly installed in individual gas exchange cuvettes and placed in a growth cabinet held at the same temperature as the growth chambers. Three replicate measurements of CO2 and δ13C entering and leaving the shoot and root compartments of each cuvette were taken every 45 min during the following 5 h. Each δ13C measurement was compared against a working standard gas, which was previously calibrated against a VPDB-gauged laboratory CO2 standard. The average standard deviation of repeated single measurements was 0.08‰ for δ13C and 0.33 µL L−1 for [CO2].
Thus, this system permitted repeated sequential measurements of the rates and isotopic composition of dark respiration by shoots and roots of individual plants, as detailed in Lehmeier et al. (2008). Rates and δ13C of shoot respiration reached constant values ∼30 min after removing plants form the stands. However, it took ∼1.5 h to completely purge the root compartment from extraneous CO2 (cf. Lötscher et al. 2004). During the 5 h measurements, dark respiration rates of roots decreased by about 3% and 6% for plants grown at low and high nitrogen supply, respectively, while that of shoots was constant in both treatments. Average rates were taken to calculate specific respiration rates (for the stability of δ13C in respired CO2 see further discussion).
Plant harvest and elemental analysis
Immediately after respiration measurements, plants were removed from the pots, washed free of sand, dissected into shoot and root, weighed, frozen in liquid nitrogen and stored at −30 °C in chest freezers. All samples were freeze-dried for 72 h, weighed again and ground to flour mesh quality in a ball mill. Aliquots of 0.75 mg ± 0.05 mg of each sample were weighed into tin cups (IVA Analysentechnik e.K., Meerbusch, Germany) and combusted in an elemental analyser (Carlo Erba NA 1110, Carlo Erba Instruments, Milan, Italy), interfaced to the CF-IRMS, to determine carbon and nitrogen contents.
Analysis of water-soluble carbohydrates
Water-soluble carbohydrates in plant biomass were analysed using a similar procedure as Thome & Kühbauch (1985). In short, 60 and 80 mg of freeze-dried ground material of shoot and root samples, respectively, were weighed in Eppendorf tubes and extracted with 2 mL H2O for 10 min in a water bath at 93 °C. Afterwards, samples were transferred to a rotating Heidolph shaker for 45 min at room temperature and then centrifuged at 20.000 g for 15 min. A 0.2 mL aliquot of the supernatant was transferred to a preparative HPLC system for separation of water-soluble carbohydrate fractions. Separation occurred in a Shodex KS 2002 chromatographic column (Showa Denko, Tokyo, Japan) held at 50 °C and a system pressure of 17 bar and at an elution rate of 0.9 mL min−1 using HPLC-grade water (Baker, Deventer, The Netherlands) as the eluent.
Samples eluting from the HPLC-system were immediately conveyed to a continuous-flow system. There, 1.25% (v/v) sulphuric acid was added at a rate of 0.9 mL min−1, and di- and oligosaccharides were hydrolysed during the ∼15 min passage through a sample loop of 12.5 m length which was kept in a water bath at 95 °C. Carbohydrates were quantified by measuring the reducing power of the hydrolysed carbohydrates by way of the oxidation-reduction reaction with potassium ferricyanide (Suzuki 1971) and detection of the reduced potassium ferricyanide solution at a wave length of 425 nm in a spectral photometer (K-2500/A4080, Knauer, Berlin, Germany). Analytical grade fructose (D(-)-Fructose, Merck, Darmstadt, Germany) served as the routine standard for carbohydrate quantification. Periodic verification with glucose, sucrose and fructan standards demonstrated relative response factors which were reasonably close to theoretical expectations: glucose, 0.94; fructose, 1.0; sucrose, 1.06; fructan, 1.04. The concentration of carbohydrates in total plant tissue was calculated from the carbohydrate contents of shoot and root samples and the shoot- and root-mass fractions of the individual plants.
The proportion of carbon in shoot- and root-respired CO2 assimilated before (unlabelled) and during labelling, funlabelled-C and flabelled-C (where flabelled-C = 1 − funlabelled-C), was obtained by means of an isotopic mass balance:
with δ13CS is the δ13C of respiratory CO2 produced by the labelled sample plant, and δ13Cold and δ13Cnew the δ13C of respiratory CO2 produced by non-labelled plants growing continuously in the chamber of origin (‘old’) or in the labelling chamber (‘new’).
For shoots, δ13CS, δ13Cold and δ13Cnew were obtained as:
where X stands for ‘sample’, ‘new’ or ‘old’ (as appropriate), and δ13Cin, δ13Cout, Fin and Fout are the isotopic compositions and the flow rates of the CO2 entering and leaving the shoot compartment, respectively. The same procedure was followed for roots. In this case, δ13Cin and Fin represented the δ13C and the flow rates of the CO2 leaving the shoot compartment (cf. Klumpp et al. 2005).
The δ13C of respired CO2 of both labelled and non-labelled (control) plants did not show a trend during the respiration measurements (which stabilized 30 min after the insertion of shoots and 1.5 h after insertion of roots in the gas-exchange cuvettes). Thus, we used the mean of all 5 h measurements of one plant to estimate funlabelled-C. This was true, except for high-nitrogen plants labelled for 1 h, where funlabelled-C of the shoot increased significantly during the 5 h measurements. In that case, funlabelled-C was taken as the y-intercept at 0 h of a linear regression of funlabelled-C (y) versus time during the 5 h measurement (cf. Fig. 5 in Lehmeier et al. 2008).
Figure 5. Sensitivity of the goodness of model fits for perennial ryegrass plants grown with a nitrogen supply of either 1.0 mm nitrate (dashed lines) or 7.5 mm nitrate (solid lines) to departures from optimized values of pool size, half-life and contribution to respiration for the pools Q1 (a, b, c), Q2 (d, e, f) and Q3 (g, h, i). Sensitivity is expressed as the root mean squared error (RMSE) of the fit (minimum value indicates the optimum value of a model parameter).
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The fraction of unlabelled carbon in CO2 respired by a plant was calculated as the flux-weighted mean of shoot and root respiration:
where Rshoot and Rroot are the absolute respiration rates (in g C h−1) of shoot and root, respectively.
Compartmental analysis of tracer time courses in respired CO2
The labelling kinetics of CO2 respired by shoots, roots and whole plants of both nitrogen treatments show that tracer incorporation occurred in distinct phases, which reflected the operation of substrate pools supplying carbon to respiration. The fitting of exponential decay functions to the tracer kinetics (similar to Moorby & Jarman 1975) indicated that the respiratory supply system included three pools in each nitrogen treatment. Yet, we also considered other compartmental concepts of respiratory carbon metabolism (e.g. Farrar 1990; Dewar, Medlyn & McMurtrie 1998) and designed several compartmental models which differed in the number of pools (two-, three- and four-pool models) or in the way the pools were interconnected and exchanged carbon with the environment (i.e. photosynthetic and respiratory fluxes).
Each model was translated into a set of equations, which described a given respiratory carbon supply system in terms of fluxes between pools and the environment using the assumption, that pool sizes were steady and fluxes obeyed first-order kinetics. Each model was tested for its ability to predict the tracer kinetics as detailed in the following for the three-pool model shown in Fig. 1. On the basis of this comparison (and respecting the principle of parsimony) the three-pool model of Fig. 1 emerged as the one closest to the real properties of the respiratory supply systems of the plants of both nitrogen supply levels. To account for a stable degree of labelling from about 2–4 h of labelling duration a delay was inserted in the model that implied that tracer release via Q1 occurred immediately after the start of labelling, while tracer release via Q2 only occurred after the delay.
The fraction of tracer in each compartment with respect to time was given by:
- ( (4a) )
where Q1, Q2 and Q3 are pool sizes and FIn is the flux of assimilated carbon (tracer) that enters the respiratory system. As the system was considered steady, FIn equalled the specific respiration rate (cf. Table 1), and FIn = FOut, FOut = F10 + F20, F12 = F20 and F23 = F32 (indices refer to donor and receptor pools, respectively, index 0 represents the environment; Fig. 1). The measured parameter against which the model prediction was compared is funlabelled-C. funlabelled-C-Qi is the fraction of unlabeled carbon in pool Qi. flabelled-C is the constant fraction of fully labelled carbon entering the system after the start of labelling. Subscript t denotes time after the onset of labelling (i.e. labelling duration), t0 refers to time just before the onset of labelling. Δt is the time step with which the model was run in the calculation and was set to 6 min. Thus, the time step was small even in comparison with the minimum time resolution of labelling, which was 1 h.
Table 1. Growth parameters of perennial ryegrass grown with either a low (1.0 mm) or a high (7.5 mm) supply of nitrate-nitrogen
|Parameter||Low nitrogen||High nitrogen|
|Specific respiration rate|| || |
| plant, mg plant-respired C g−1 plant-C h−1||0.99 ± 0.03||1.50 ± 0.02|
| shoot, mg shoot-respired C g−1 plant-C h−1||0.62 ± 0.02||0.97 ± 0.02|
| root, mg root-respired C g−1 plant-C h−1||0.37 ± 0.01||0.53 ± 0.01|
|Specific growth rate, mg C g−1 C h−1||1.58 ± 0.31||3.23 ± 0.21|
|Specific nitrogen uptake rate, mg N g−1 C h−1||0.035 ± 0.007||0.124 ± 0.010|
|Shoot : root ratio||2.96 ± 0.10||3.84 ± 0.14|
|C : N ratio (w/w)||48.8 ± 1.3||24.1 ± 0.5|
The set of equations (4) was implemented in a custom-made program using the free software ‘R’ (R Development Core Team 2007). Initial values for pool sizes, fluxes between pools and the delay were inserted, and the equations were solved. In that way, a tracer time course across the entire labelling period (600 and 696 h for plants grown at high and low nitrogen supply, respectively) was generated. The quality of the fit was expressed as the root mean squared error (RMSE).
This procedure was executed millions of times by stepwise and systematic variation of preset values for pool sizes, fluxes between pools and the delay. In doing so, the combinations of pool sizes, fluxes and the delay giving the best fits, i.e. the lowest RMSEs, were taken as the ones closest to the real properties of the respiratory supply systems. This extensive scanning procedure aimed to detect the global minimum RMSE rather than a local minimum, an aspect, compartmental analyses must cope with. This procedure also revealed the sensitivity of the fits to changes in parameter values. The minimum RMSE of each RMSE response curve corresponds to the optimum of pool half-lives, sizes and contributions. A high sensitivity of the goodness of fit to changes in one parameter is revealed by steep increases of the RMSE response curve on either side of the optimum.
Optimized pool sizes and fluxes served to calculate the half-life of a pool of size Qi:
with Fi the sum of all fluxes leaving the pool Qi.
The quantitative contribution of a pool Qi (CQi) to respiratory carbon release was derived based on optimized fluxes. It is defined here as the probability of tracer moving in a certain flux of the respiratory system (Fig. 1):
CQ1 is the probability that tracer enters the system and leaves it in F10 without visiting any other pool. CQ2 implies, that tracer enters Q2 via Q1 and is respired in F20 without moving through Q3. CQ3 is the probability of tracer cycling through Q3 at least once.
The mean residence time of carbon in the respiratory supply system (τ) was calculated as
with Qtotal the total size of all respiratory substrate pools, i.e. the sum of Q1, Q2 and Q3 in mg C g−1 plant-C (Table 2) and rplant the specific respiration rate of the whole plant (Table 1) in mg C g−1 plant-C h−1.
Table 2. Optimized parameters of the model shown in Fig. 1 as fitted to tracer time courses of CO2 respired by perennial ryegrass plants (Fig. 4c) grown with either a low (1.0 mm) or a high (7.5 mm) supply of nitrate-nitrogen
| ||Low nitrogen||High nitrogen|
| ||Size (mg C g−1 plant-C)|
| ||Half-life (h)|
| ||Contribution (%)|
| ||Flux (mg C g−1 plant-C h−1)|
| ||Delay (h)|
The analysis of tracer time courses conducted in the present study holds the assumptions generally made in compartmental modelling, namely: (1) the system is in a steady-state, i.e. pool sizes and fluxes in the system are constant and only funlabelled-C in respired CO2 changes with time; (2) fluxes obey first-order kinetics; and (3) pools are homogeneous and well mixed. Support for the validity of assumption (1) is obtained by the constancy of plant specific growth and respiration rates (Figs 2 & 3). Plant growth in continuous light eliminated short-term changes in pool sizes and fluxes which would have complicated the analysis in day/night cycles. Assumption (2) is probably false in a strict sense, but support for its practical validity has been found repeatedly (see Farrar 1990 for a discussion). Assumption (3) is a simplification, particularly for studies at the whole organ and plant level, in the sense that different pools are probably not biochemically homogeneous and distributed in different tissues. Yet, in the context of labelling, a pool is defined as a set of compounds which exhibit the same proportion of labelled carbon atoms. So, in principle, one pool can include several populations of anatomical (physical) features and biochemical species on the condition that they exhibit the same proportion of label (Atkins 1969; Rescigno 2001).
Figure 2. Total carbon mass of perennial ryegrass grown with a nitrogen supply of either 1.0 mm nitrate (open symbols) or 7.5 mm nitrate (closed symbols). Each value is the mean of 3–6 replicate plants. Lines denote linear regression (P < 0.05; see also Table 1). Note the logarithmic scaling of the y axis.
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Figure 3. Specific respiration rates of perennial ryegrass grown with a nitrogen supply of either 1.0 mm nitrate (open symbols) or 7.5 mm nitrate (closed symbols), labelled for different time intervals, and of non-labelled controls (C; at left). Each value is the mean of 3–12 replicate plants (±1SE). Average rates were 46.3 ± 0.8 (n = 56) and 35.7 ± 0.7 (n = 60) for low- and high-nitrogen plants, respectively (dashed lines). Regression analysis yielded no significant trends (P > 0.05). Note the logarithmic scaling of the x axis.
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