Supporting Information to “Daylength effects on carbon stores for
respiration of perennial ryegrass” by Lehmeier et al. (2010) Methods S1
and Table S1
Methods S1
Plant material and growth conditions
Lolium perenne L. cv. Acento was grown in controlled environments using the mesocosm
13
CO2/12CO2 gas exchange and labelling facility described by Schnyder et al. (2003). Seeds
were sown individually in plastic pots (350 mm high, 50 mm diameter) which were filled with
800 g of washed quartz sand (0.3–0.8 mm grain size). Pots were arranged in growth chambers
(Conviron E15, Conviron, Winnipeg, Canada) with stand densities of 378 plants m-2. Every
three hours, the plants received a modified Hoagland-type nutrient solution containing 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, 0.125 mM iron as EDTA, and micronutrients. Stands
were periodically flushed with demineralised water to prevent salt accumulation. In the
growth chambers, temperature was held constant at 20 ºC and relative humidity near 85 %.
Light was supplied by cool-white fluorescent lamps.
Stands in two growth chambers experienced continuous illumination with a photosynthetic
photon flux density (PPFD) of 275 µmol m-2 s-1 at canopy height. Four growth chambers were
operated with day/night cycles with alternating 16 h photoperiod (PPFD 425 µmol m-2 s-1) and
8 h darkness periods. Thus, plants of both light treatments received the same total daily
photon flux density (24 mol photons m-2 d-1). PPFD was measured with a quantum sensor (Li190SZ, Li-Cor Inc., Lincoln, USA).
CO2 control in the growth chambers
Air supply to the growth chambers was generated by mixing CO2-free air with CO2 of known
carbon isotope composition (δ13C = [(13C/12C)sample / (13C/12C)VPDB standard] – 1). The CO2
concentration during illumination was held constant at 360 µL L-1 inside each chamber, and
was constantly monitored by an infrared gas analyzer (Li-6262, Li-Cor Inc.). The δ13C in the
chambers’ atmospheres was measured online with a continuous-flow isotope-ratio mass
spectrometer (Delta plus, Finnigan MAT, Bremen, Germany). The rate of CO2 supply to the
chambers exceeded the CO2 exchange rate of the stands in light by a factor of 9. This
minimized effects of photosynthesis, respiration and recycling of respired CO2 on the δ13C of
chamber CO2. Contamination of chamber CO2 during plant handling was minimized by the
use of custom-made air-locks (see Lehmeier et al., 2008).
13C
labelling
Plants were grown in atmospheres with constant δ13C of CO2 which originated either from a
fossil-organic, 13C-depleted or from a mineral, 13C-enriched source. Plants were labelled by
changing the δ13C of the atmospheric CO2 to which they were exposed to (i.e. 13C-enriched →
13
C-depleted atmosphere or vice versa) and thus, of the photosynthetically fixed carbon. As
described in detail by Lehmeier et al. (2008), the difference in δ13C of the CO2 was ~26 ‰ in
the continuous light treatment. These plants were labelled for periods ranging from 1 h to 25
d, followed by immediate respiration measurements (see below). The average plant age at the
time of respiration measurements was 48 d.
In the day/night treatment, stands in two chambers received CO2 with δ13C of 0.3 ‰,
while the two other chambers received CO2 with δ13C of -42.2 ‰ (all CO2 from Linde AG,
Höllriegelskreuth, Germany). That is, δ13C of photosynthetically fixed carbon differed by ~42
‰. Six weeks after sowing, the source CO2 for the chambers was switched from 13C-enriched
CO2 to 13C-depleted CO2 or vice versa. The switch occurred two hours before the end of a
regular dark period, ensuring complete replacement of chamber CO2 by the new CO2 at the
beginning of the next light period (1st labelling day). Respiration measurements were
performed after 2, 4, 8, 12 or 16 h of labelling (1st light period following the switch of CO2),
after 32 h and 40 h (middle and end of the 2nd light period), and at the end of the 3rd, 4th, 6th,
8th, 12th and 16th light period in the presence of the labelling CO2. The average age at
measurements was 45 d for these plants.
Stand scale ‘online’ carbon isotope discrimination (Δ) during gas exchange in light
prior to the onset of labelling was frequently determined in all growth chambers according to
Evans et al. (1986). Within treatments, Δ did not differ between growth chambers with
different δ13C in chamber air CO2 (P>0.05), demonstrating that any contamination with
extraneous CO2 was insignificant and did not perturb the labelling (Schnyder, 1992).
Respiration measurements
Rates of shoot and root respiration, and the δ13C of shoot- and root-respired CO2 of individual
labelled and non-labelled (control) plants were measured in the gas-exchange system
described by Lötscher et al. (2004) and Klumpp et al. (2005). Plants were removed from the
stands at the end of the labelling intervals, rapidly installed in individual gas exchange
cuvettes and placed in a growth cabinet held at the same temperature as the growth chambers
(Lehmeier et al., 2008). Three replicate measurements of the concentration and δ13C of CO2
entering and leaving the shoot and root compartments of each cuvette were taken every 45
min during 5 h (plants in continuous light) or 6 to 7 h (day/night cycle). Each δ13CCO2
measurement was compared against a working standard gas which was previously gauged
against a VPDB-calibrated laboratory CO2 standard. The average standard deviation of
repeated single δ13C measurements was 0.10 ‰ for continuous light and 0.05 ‰ for the
day/night treatment. The standard deviation of δ13C of respired CO2 of replicate non-labelled
(control) plants was 0.9 ‰ for shoots and 0.7 ‰ for roots in continuous light, and 0.7 ‰ and
1.1 ‰ for shoots and roots of plants in day/night cycles. This was less than 4 % of the label
signal (see above).
Generally, reliable measurements of the rate and δ13C of shoot respiration were
obtained approximately 30 min after transfer from the stand. However, it took up to 1.5 h to
completely purge the root compartments from extraneous CO2 (see Lötscher et al., 2004). In
continuous light, the specific respiration rate of roots decreased by about 6 % during the 5 h
measurement cycle, but that of shoots was constant. In day/night cycles, shoot respiration rate
decreased by 4 % during the dark period, but no change was observed for root respiration
rates. In both cases, specific respiration rates were independent of the time of removal from
the chambers (P>0.05).
Plant harvest and elemental analysis
Immediately after respiration measurements, plants were removed from the pots and the sand
was washed off the roots. The plants were dissected into shoot and root, weighed, frozen in
liquid nitrogen, and stored at -30 °C. 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). The tin cups
were combusted in an elemental analyzer (Carlo Erba NA 1110, Carlo Erba Instruments,
Milan, Italy) to determine carbon and nitrogen elemental contents.
Analysis of water-soluble carbohydrates
Water-soluble carbohydrate fractions of fructan, sucrose, glucose and fructose in plant
biomass were extracted and quantified as described by Lehmeier et al. (2010). The analysis
was performed separately for shoot and root samples of plants that were used for respiration
measurements. Thus, these plants had experienced 5-6 h of darkness after removal from the
growth chambers in the continuous light treatments, and 6-7 h in the day/night treatment.
Calculation of tracer time course in respired CO2
The proportion of carbon in shoot- and root-respired CO2 that was assimilated before labelling
(funlabelled-C) and during labelling (flabelled-C, where flabelled-C = 1 – funlabelled-C) was calculated by
isotopic mass balance:
(1)
funlabelled-C = (δ13CS – δ13Cnew) / (δ13Cold – δ13Cnew)
where δ13CS, δ13Cold and δ13Cnew are the δ13C of the CO2 respired by a labelled sample plant
(‘S’), and by plants which were continuously exposed to unlabelled (‘old’) or labelling
(‘new’) CO2. Thus, δ13Cold and δ13Cnew represent the unlabelled and completely labelled end
members of the mass balance, respectively.
For the shoots, δ13CS, δ13Cold and δ13Cnew were obtained as
(2)
δ13CX= (δ13Cin Fin – δ13Cout Fout) / (Fin – Fout),
where X stands for ‘S’, ‘new’ or ‘old’ (as appropriate); δ13Cin, δ13Cout, Fin and Fout are the
isotopic signatures and flow rates of the CO2 entering and leaving the shoot cuvette,
respectively. Calculations for the root compartment were done in the same way considering
that the concentration and δ13C of the CO2 entering the root compartment was equal to that
measured in the shoot outlet (compare with Klumpp et al., 2005).
The δ13C of shoot- and root-respired CO2 of non-labelled as well as labelled plants of both
light treatments showed no trend during the course of the respiration measurements (P>0.05),
except for one case, which was accounted for in the calculations (see Lehmeier et al., 2008;
2010). These findings are in agreement with those of Werner et al. (2009) for a fast-growing
herb who observed no significant changes in δ13C of leaf-respired CO2 during the dark period.
However, any rapid change in δ13C of respired CO2 which may have occurred shortly after the
light-to-dark-transition (Barbour et al., 2007) would not have been detected in our experiment
since the first measurement was only obtained 30 minutes after moving the plants from light
to dark conditions (see above).
Compartmental analysis of tracer time course
Functional and structural information about the substrate supply system of respiration can be
extracted from the tracer time course (the evolution of funlabelled-C in respired CO2 with time;
Fig. 1) using compartmental analysis (Lehmeier et al., 2008). This information includes the
number of pools in the supply system, the topology of the system, the size and half-life of
each pool, and the fractional contribution of current assimilates and stores to shoot and root
respiration.
Such a compartmental analysis is straightforward for plants in continuous light, which
exhibited near-constant physiological features over time scales of hours to several days
(Lehmeier et al., 2008; 2010). However, plants grown in day/night cycles exhibit nonconstant physiological activities at timescales of less than one day. An obvious example of
such a change is the discontinuity of photosynthesis in day/night cycles and (possible)
consequences for metabolite pool sizes and fluxes. This complicates the application of a
compartmental model to the tracer kinetics. We overcame this difficulty by restricting the
compartmental analysis to the day-by-day scale (Lattanzi et al., 2005) in both treatments. On
that time-scale, relevant physiological features such as shoot and root specific respiration rates
and nitrogen concentration in shoot and root biomass (Table 1) were near-steady, that is they
did not change significantly with plant age during the observed time (P>0.05; data not
shown).
For plants grown in continuous light, we calculated the mean funlabelled-C in CO2
respired during a 1 day-long period (or multiples of that, depending on the time resolution of
the data) by linear interpolation between the actual measurements. From this, we calculated
the daily mean funlabelled-C in respired CO2 on the 1st and 2nd labelling day, and means for the
intervals of days 3 to 4, 5 to 8, 9 to 12, 13 to 17 and 18 to 25.
For plants grown in day/night cycles, funlabelled-C in CO2 respired in each light period
was also derived by linear interpolation. funlabelled-C in CO2 respired by these plants during the
regular dark periods was constant within each dark period (see above). The mean funlabelled-C in
CO2 respired during the 1st, 2nd, 3rd, 4th labelling day, and during the intervals of days 5 to 6, 7
to 8, 9 to 12, and 13 to 16 of continuous labelling was then calculated as the respirationweighted average of funlabelled-C in the light and dark period of a given day (or interval of
several days).
For plants grown in day/night cycles, we considered that the rate of shoot respiration
in light is somewhat lower than that measured in darkness (e.g. Pärnik et al., 2007), and that
the degree of this light inhibition potentially affected the relative contributions of products of
current assimilation and stores to respiration. We therefore calculated the respiration-weighted
average of funlabelled-C of plants in day/night cycles assuming that shoot respiration in light was
reduced by 0 %, 30 % or 65 % relative to the measured rates of dark respiration by shoots (i.e.
Rday/Rnight being either 1, 0.7 or 0.35, respectively). This covered a range of findings for the
degree of light inhibition of respiration (e.g. Peisker & Apel, 2001; Pärnik et al., 2007). For
plants in continuous light, such assumptions were obviously not necessary.
For both treatments, the time course of the mean funlabelled-C in respired CO2 was then
subject to compartmental analysis. For this we assumed that (1) the system was in steadystate, (2) fluxes obeyed first-order kinetics, that is Fxy = kxy*Qx (Fxy, flux out of pool x into
pool y; kxy, respective rate constant; Qx, size of pool x), and (3) pools were well mixed, as
discussed before (Lattanzi et al., 2005; Lehmeier et al., 2008). This included the notion, that a
pool is defined as a set of compounds which exhibit the same fraction of labelled carbon
atoms (Atkins, 1969; Rescigno, 2001). Thus, in principle, a pool can include several
biochemical compounds, possibly even located in different physical spaces, on the condition
that they all supplied respiration and showed the same degree of labelling with time.
One-, two- and three-pool models were tested in the analysis. The models were
defined as sets of differential equations describing the fluxes between pools and with the
environment (see eqs. 3). The models were implemented in the software ModelMaker
(Cherwell Scientific, Oxford, UK) and the differential equations solved using the 4th order
Runge-Kutta numerical method. Levenberg-Marquardt optimization was used to find model
parameters that minimized the sum of the squared differences between model’s predictions
and observed tracer kinetics. We furthermore followed a previously described approach
(Lehmeier et al., 2008) where a custom-made computer program systematically tested a large
number of parameter-combinations. The visualization of these results helped to ensure that
global rather than local best fits were detected. The principle of parsimony and the extra sumof-squares F-test for comparing models as described by Motulsky & Christopoulos (2004)
guided the selection process. This resulted in the two-pool model in Figure 2 as the simplest
and biologically most meaningful model which was able to describe the observed tracer time
courses.
For this model, the fraction of tracer in the pools Q1 and Q2 with respect to time was
given by:
(3a)
dQ1/dt = Tracer + k21*Q2 – k10*Q1 – k12*Q2
(3b)
dQ2/dt = k12*Q1 – k21*Q2
with the rate constants governing the flux from pool Q1 to Q2 (k12), that from Q2 to Q1 (k21)
and the respiratory flux out of Q1 (k10). Tracer represents the import flux of labelled carbon
into the system. In the steady-state, this equals the export flux (respiration). As the time
course of funlabelled-C in respired CO2 converged to an asymptotic value significantly larger than
zero (see ‘Tracer time course in respired CO2’ in the Results section), Tracer is given by:
(4)
Tracer = respiration rate * (1 – asymptote),
where respiration rate was given by the measured specific respiration rates of the plants
(Table 1). ‘asymptote’ represents the fraction of respiration supplied by a substrate source that
did not release any detectable amount of tracer during the entire labelling period.
In the steady-state, that is, dQ1/dt = dQ2/dt = 0, pool sizes were given by
(5a)
Q1 = Tracer/k10
(5b)
Q2 = Tracer/k10 * k12/k21
The rate constants kxy and the asymptote were the optimized parameters. The half-life t1/2(Qx)
of a pool Qx was calculated from the rate constants according to:
(6)
t1/2(Qx) = ln(2)/kx
with kx the sum of all rate constants kxy leaving the pool Qx. The fractional contribution of
current assimilates to respiration is defined as the probability that tracer leaves the system
without cycling through Q2 (i.e. current assimilate that was not stored), while the fractional
contribution of stores to respiration is the probability that tracer cycles through Q2 at least
once before it is respired (i.e. that tracer underwent storage). These fractional contributions
are given by:
(7a)
Contribution of current assimilates = k10/(k10+k12)
(7b)
Contribution of temporary stores = k12/(k10+k12)
The sum of the contribution of current assimilates, stores and the asymptote-value equals 1.
References
Atkins GL. 1969. Multicompartment models in biological systems. London, UK: Methuen.
Barbour MM, McDowell NG, Tcherkez G, Bickford CP, Hanson DT. 2007. A new
measurement technique reveals rapid post-illumination changes in the carbon isotope
composition of leaf-respired CO2. Plant, Cell & Environment 30: 469-482.
Evans JR, Sharkey TD, Berry JA, Farquhar GD. 1986. Carbon isotope discrimination
measured concurrently with gas-exchange to investigate CO2 diffusion in leaves of
higher plants. Australian Journal of Plant Physiology 13: 281-292.
Klumpp K, Schäufele R, Lötscher M, Lattanzi FA, Feneis W, Schnyder H. 2005. Cisotope composition of CO2 respired by shoots and roots: fractionation during dark
respiration? Plant, Cell & Environment 28: 241-250.
Lattanzi FA, Schnyder H, Thornton B. 2005. The sources of carbon and nitrogen supplying
leaf growth: assessment of the role of stores with compartmental models. Plant
Physiology 137: 383-395.
Lehmeier CA, Lattanzi FA, Schäufele R, Wild M, Schnyder H. 2008. Root and shoot
respiration of perennial ryegrass are supplied by the same substrate pools: assessment by
dynamic
13
C labeling and compartmental analysis of tracer kinetics. Plant Physiology
148: 1148-1158.
Lehmeier CA, Lattanzi FA, Schäufele R, Schnyder H. 2010. Nitrogen deficiency increases
the residence time of respiratory carbon in the respiratory substrate supply system of
perennial ryegrass. Plant, Cell & Environment 33: 76-87.
Lötscher M, Klumpp K, Schnyder H. 2004. Growth and maintenance respiration for
individual plants in hierarchically structured canopies of Medicago sativa and
Helianthus annuus: the contribution of current and old assimilates. New Phytologist
164: 305-316.
Motulsky H, Christopoulos A. 2004. Fitting models to biological data using linear and
nonlinear regression. New York, US: Oxford University Press.
Pärnik T, Ivanova H, Keerberg O. 2007. Photorespiratory and respiratory decarboxylations
in leaves of C3 plants under different CO2 concentrations and irradiances. Plant, Cell &
Environment 30: 1535-1544.
Peisker M, Apel H. 2001. Inhibition by light of CO2 evolution from dark respiration:
Comparison of two gas exchange methods. Photosynthesis Research 70: 291-298.
Rescigno A. 2001. The rise and fall of compartmental analysis. Pharmacological Research
44: 337-342.
Schnyder H. 1992. Long-term steady-state labelling of wheat plants by use of natural
13
CO2/12CO2 mixtures in an open, rapidly turned-over system. Planta 187: 128-135.
Schnyder H, Schäufele R, Lötscher M, Gebbing T. 2003. Disentangling CO2 fluxes: direct
measurements of mesocosm-scale natural abundance
13
CO2/12CO2 gas exchange,
13
C
discrimination, and labelling of CO2 exchange flux components in controlled
environments. Plant, Cell and Environment 26: 1863-1874.
Werner C, Wegener F, Unger S, Nogués S, Priault P. 2009. Short-term dynamics of
isotopic composition of leaf-respired CO2 upon darkening: measurements and
implications. Rapid Communications in Mass Spectrometry 23: 2428-2438.
Table S1 Optimized parameters of the two-pool compartmental model of respiratory
substrates as shown in Figure 2, applied to the mean fraction of unlabeled carbon in CO2,
respired by shoots of plants grown in a 16/8 h day/night regime, under the assumption that
there was either no inhibition of shoot dark respiration rate in light (Rday/Rnight = 1) or that the
shoot respiration rate in the light was 0.35 times the shoot respiration rate in the dark. The
model is provided with an asymptote which represents a source of respired carbon that
released no tracer during the entire labelling period. Values ± 95 % CI were calculated from
model-optimized rate constants (and the measured respiration rate for the size estimation).
Rday/Rnight = 1
Rday/Rnight = 0.35
size (mg C g-1 plant C)
Q1
23 ± 4
19 ± 4
Q2
29 ± 19
30 ± 18
half-life (h)
Q1
5.8 ± 2.0
5.2 ± 1.6
Q2
12 ± 3
14 ± 4
fractional contribution (%)
current assimilates
38 ± 13
40 ± 12
temporary stores
56 ± 13
53 ± 12
asymptote
6±1
6±1