Plant material and growth conditions
Plants of sunflower (Helianthus annuus L., cv. Optisol, CARG, BSA-Nr. 241) were sown individually in plastic pots (35 cm deep, 5 cm diameter), filled with 0.8 kg of washed quartz sand (0.4–0.8 mm particle size), transferred to four growth cabinets (Conviron E15, Conviron, Winnipeg, Canada) and arranged at a density of 78 plants m−2. Throughout the experiment an automatic drip irrigation system supplied nutrient solution [modified half-strength Hoagland solution with 7.5 mM N L−1; composition: 2.5 mM KNO3, 2.5 mM Ca(NO3)2, 1 mM MgSO4, 0.5 mM KH2PO4, 0.1 mM Fe–ethylenediaminetetraacetic acid (EDTA), 23 µM H3BO3, 4.6 µM MnCl2, 0.4 µM ZnSO4, 0.16 µM CuSO4, 0.26 µM H2Mo8O4] to every pot every 4 h. The supply rate was adjusted weekly to satisfy the water demand of plants. Light was supplied by cool white fluorescent tubes and incandescent lamps. Irradiance at the top of each canopy was maintained at 600 µmol m−2 s−1 photosynthetic photon flux density (PPFD) during the 16 h photoperiod. Temperature was controlled at 20/16 °C and relative humidity at 75/85% during the light and dark periods of the day. At 28 d after sowing ABA (Sigma-Aldrich, Seelze, Germany) dissolved in methanol was added to the nutrient solution of two growth cabinets. The final ABA concentration in the nutrient solution was 20 µM L−1.
Gas exchange measurements and isotope analysis
The four growth cabinets formed part of the 13CO2/12CO2 gas exchange and labeling system described by Schnyder et al. (2003). In short, air supply to the growth cabinets was generated by mixing CO2- and H2O-free air with CO2 of known carbon isotope composition (δ13C, where δ13C = [(Rsample/Rstandard) − 1], and R the 13C/12C ratio in the sample or Vienna Peedee belemnite (VPDB) standard. The CO2-free dry air was produced by passing compressed ambient air through an absorption dryer (KEN 3100, Zander, Essen, Germany) filled with a molecular sieve (activated aluminium oxide F200, Alcoa, Houston, TX, USA). The dryer was fed by a screw compressor (S40, Boge, Bielefeld, Germany), which compressed ambient air to ∼0.7 MPa at a rate of ≤300 m3 h−1. After passage through the dryer, the air had a residual CO2 concentration of <0.5 µmol mol−1 and its dew point was ≤70 °C. The rate of air supply and the CO2 concentration in air injected into the growth cabinets was controlled by four independently operated gas mixing systems. Each served one cabinet and comprised two computer-operated mass-flow controllers (FC-2925V for air and FC-2900 4S for CO2, Tylan General, San Diego, CA, USA). A computer-controlled gas sampling system (DMP, Hegnau-Volketswil, Switzerland) sequentially sampled air entering and leaving each growth cabinet. Each air sample was split: CO2- and H2O-concentration was analysed with an IRGA (Licor 6262, Li-Cor, Lincoln, NE, USA) and the δ13C of CO2 by an online gas isotope ratio mass spectrometer (IRMS, Delta plus, Finnigan MAT, Bremen, Germany). Air for mass spectrometric analysis was pumped continuously via a steel capillary from the gas sampling system to a 300 µL sample loop which was attached to a six-port Valco valve (Valco Instruments Co. Inc., Houston, TX, USA) mounted in a GC interface (GP-GC Interface, Finnigan MAT, Bremen, Germany). Air samples were fed to the mass spectrometer by periodically flushing the sample loop with He carrier gas and flushing the content through a Nafion® water trap and a GC column (25 m × 0.32 mm Poraplot Q, Chrompack, Middelburg, Netherlands). Then, the CO2 was introduced into the ion source of the IRMS via a glass capillary (0.1 mm i.d.) connected to the interface via an open split.
The system allowed a high frequency of sampling (approx. one sample per 2 min), so that each inlet and outlet of the four growth cabinets was sampled every 25 min. Each sample was compared with a VPDB-gauged working standard reference CO2 gas. The external precision (standard deviation) at 300 µmol mol−1 CO2 was <0.2‰ for δ13C.
During the entire experiment, including the establishment phase of the stands, two of the growth cabinets were supplied with CO2 from a fossil-organic source (δ13C −47.1‰) and two with CO2 from a mineral source (δ13C −5.4‰; both CO2: Messer Griesheim, Frankfurt a.M., Germany).
Rates of net CO2 exchange in light (A, µmol CO2 m−2 s−1) were calculated as the difference between the CO2 fluxes entering (Fin, µmol CO2 s−1) and leaving (Fout, µmol CO2 s−1) the cabinets divided by the cabinets' ground area (s, 1.4 m2):
δA, the δ13C of net CO2 exchange of canopies during the light was obtained by isotopic mass balance:
with subscript in and out denoting the inlet and outlet of the growth cabinet. δRn, the δ13C of CO2 respired in the dark was obtained in the same way using measurements taken during the dark period.
with and C the CO2 concentration at standard humidity.
Cout was maintained at 310 µmol mol−1 (SE = 0.6 µmol mol−1) throughout the experiment. Cin and the flux of air into the chamber were adjusted at intervals of 2 to 3 d to maintain ξ between 5 and 8. Thus, with constant Δobs during canopy development, δ13C of the CO2 in the cabinet atmosphere (δout) was constant (SE = 0.04‰ for both CO2) throughout the experiment.
Transpiration rate (E, mmol H2O m−2 s−1) was calculated analogous to A (Eqn 1). Thus, E = (FH2O out − FH2O in)/s, with out designating the flux of water vapour leaving the cabinet (mmol H2O s−1) and in that added to cabinet air by the individual cabinets' humidification system (note that air entering the chambers was dry; see earlier discussion). The humidification system controlled the water vapour concentration in cabinet air by adding just enough vapour to match the difference between that generated by E and the nominal water vapour concentration of the chamber. Water addition by the humidification system occurred by activation (opening) of spray nozzles whenever the measured vapour concentration decreased below the nominal concentration. The rate of vapour addition of each sprayer was obtained from a calibration of vapour production versus nozzle activation times. E was recorded for each step during CO2 response measurements for approximately 15 min when the CO2 concentration at the chamber outlet had reached the new steady state. Evaporation from the sand surface (15% of chamber area) was a negligible component of (evapo)transpiration of the closed sunflower canopy. This was true, except for a small evaporation signal during a short period during and after irrigation events when the sand surface of pots was wetted and the relative humidity of chamber increased briefly. Therefore, data were not taken for a period of 20 min after irrigation. The CO2 response of A and E was measured in duplicate in two growth cabinets.
Calculation of gm
According to Fick's first law, gm is expressed as,
with A the net photosynthesis rate of the stand and Ci and Cc the CO2 concentrations in the intercellular airspace and in the chloroplast. Canopy-scale on-line carbon isotope discrimination (Δobs) is (Tcherkez et al. 2010):
with Ca and Cs denoting the CO2 concentration in the atmosphere and at the leaf surface, ab the fractionation during diffusion through the leaf boundary layer; a that during diffusion in air, am the combined fractionation during dissolution of CO2 in water and fractionation during diffusion of dissolved CO2 in water; b the fractionation associated with carboxylations, Rd the rate of leaf dark respiration in light; Rh the dark respiration of heterotrophic plant parts (root and stem), k the carboxylation efficiency, Γ* the CO2 compensation point in the absence of dark respiration, f the fractionation associated with photorespiration, e the fractionation associated with leaf dark respiration in light and eh the fractionation associated with respiration of heterotrophic plant parts. d*/Ca, the last term on the right-hand side of Eqn 5a, represents the effect of total mesocosm respiration in light on Δobs. This includes photorespiration and daytime dark respiration of autotrophic (leaf) and heterotrophic plant parts and soil, and was termed (photo)respiration (in accordance with Tcherkez et al. 2010). This term is a potentially large component of Δobs as respiration is a much bigger fraction of net CO2 exchange at mesocosm than at leaf scale, and carbon isotope fractionation in photorespiration and dark respiration of different plant parts is significant (Ghashghaie et al. 2003; Klumpp et al. 2005). The contribution of (photo)respiration to Δ, d*/Ca was obtained as:
(cf. Eqn 3 in Tcherkez et al. 2010), where Δ0 denotes Δ at the CO2 compensation point, Γ, of the mesocosm. Δ0 was estimated as the intercept of a linear regression of Δobs versus A (‘second method’ in Tcherkez et al. 2010) using the data obtained during the CO2 response measurements (as presented in Fig. 2a,c). This method has the advantage, that it does not require assumptions on the rates of (photo)respiration of leaves, and respiration of roots and stems and their specific fractionation factors (see discussion in Tcherkez et al. 2010).
Figure 2. Response of (a) A (b) E and (c) Δobs of sunflower canopies to a change in Ca, the atmospheric CO2 concentration. Measurements were performed in the absence (full symbols) and presence (open symbols) of abscisic acid (ABA) in the nutrient solution. Measurements were performed on the day just prior to ABA addition, and on the third day after ABA addition, when A and Δobs had decreased to a near-constant level. All measurements were performed in two mesocosms. Data points represent single measurements.
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Mesophyll conductance was calculated using the difference between Δobs (Eqn 3) and an estimate of Δ assuming infinite gm (Δi):
Leaf boundary layer conductance used to calculate Cs was not measured but assumed to be 750 mmol m−2 s−1 for water vapour (within the range found for broad-leafed species in greenhouses; Morrison & Gifford 1984; Aphalo & Jarvis 1993; Gan et al. 2002). Ci was calculated from stomatal conductance, which was based on measurements of transpiration (E) and leaf temperature. Leaf temperature was measured using six thermocouples evenly distributed within the top 20 cm of the canopy.
The last term in Eqn 7[i.e. the (photo)respiratory contribution] is the same as the last term in Eqn 5a. Thus, any difference between Δi and Δobs was caused by the difference between Ci and Cc:
Equation 8 could be solved for Cc as the only unknown and used to calculate gm using Eqn 1.