Controlled environment chambers
Rice plants (Oryza sativa L., cv. Nipponbare) of Japonica-type were grown in six naturally sunlit, semiclosed growth chambers for an entire growing season. Chamber dimensions were 4 × 3 × 2 m (L × W × H) with the space for plant growth 4 × 2 × 2 m. Each chamber housed two stainless-steel containers (1.5 × 1.5 × 0.3 m; L × W × D) filled with paddy soil. The frames, rear (north) walls, and floor of the chamber were made of stainless steel. The frames were glazed with 5-mm-thick tempered glass whose transmittance of visible light were > 80%. Air temperature and rh in each chamber were controlled by electrical resistive heaters (with bubbling system for humidification) and cold-water heat exchangers using PID (Proportional + Integral + Derivative) controllers (DB1000, CHINO, Tokyo, Japan). Air temperature and rh in each chamber were measured with temperature-humidity sensors (HN-Q500-1, CHINO, Tokyo, Japan) shielded against direct solar radiation and mounted above a rice canopy. In this experiment, air temperature was controlled to track ambient air temperature with the seasonal mean temperature being 23.4°C and a rh of 80 ± 1.9%. [CO2] was maintained at 353 ± 15/396 ± 23 µmol mol−1 (day/night) in three ambient [CO2] chambers and 667 ± 36/700 ± 41 µmol mol−1 (day/night) in three elevated [CO2] chambers. Daytime [CO2] was maintained by a computer-controlled pure CO2 injection system, which compensated for CO2 uptake by the rice canopy. During night-time , [CO2] increased due to plant respiration, but was kept below 100 µmol mol−1 higher than the daytime [CO2] by a computer-controlled air ventilation system, which introduced ambient air to reduce [CO2]. Ambient air temperature was measured with a platinum resistance thermometer which was shielded, aspirated and placed outside the chambers. Environmental data in each chamber and ambient air temperature were monitored every 10 s, and 5 min means were recorded. Ambient [CO2] data were provided by the laboratory of Micrometeorology of NIAES. They monitored ambient [CO2] every 10 s at several heights on an observation tower, which was located c. 50 m south of these chambers.
Germinated seeds of rice were sown on 20th April in 1998. Seedlings were grown in plug pots at 23°C, 80% rh and 350 or 650 µmol mol−1[CO2]. On 15th May, they were transplanted in the containers in chambers with 3 seedlings per hill at 20 × 20 cm spacing. Plants were fertilized with 5 g N, 15 g P2O5, and 15 g K2O per m2 just before transplanting, and 3 g N per m2 on 56 d after transplanting (DAT). The amount of fertilizer was based on local agronomic practices. The containers were flooded with water at 1–5 cm depth throughout the season. When leaf area index of rice canopy reached 3, shading nets (50% light transmittance) were installed at canopy height along the outside of each chamber to make a light environment similar to that in a field. The rice plants were harvested on 15th October (153 DAT).
Growth and yield measurement
Three rice hills were destructively sampled from each chamber at 20, 40, 67, 98, 122 and 153 DAT. The gaps of the sampled plants were filled with potted plants grown outdoors with the same nitrogen application as those in the chambers. These pot-grown plants were not included in any further sampling. After leaf area was measured for each sampled plant, plants were detached into leaf blade, leaf sheath + stem, root, ear and dead leaf blade. Then each sample was oven-dried for > 48 h at 80°C and d. wt was determined. Samples of one chamber from each [CO2] treatment were used for carbon (C) and nitrogen (N) analysis. After grinding samples, C and N concentration were determined by a CN coder (MT-700, Yanako, Kyoto, Japan). At harvest, 12 rice hills were destructively sampled from each chamber and the yield was determined for each chamber.
Canopy CO2 exchange rate
[CO2] in each chamber was monitored every 10 s by an infra-red CO2 controller (ZFP9GD11, Fuji-denki, Tokyo, Japan) and recorded every 5 min as a 5-min average. For a more precise measurement of [CO2] in the chambers than the CO2 controllers, sample air from each of the six chambers was taken to the control house, and [CO2] was determined by an infra-red gas analyser (IRA-107, Shimadzu, Kyoto, Japan), which was automatically calibrated three times a day against nitrogen (zero) gas and standard CO2 gas (700 µmol mol−1). It took 5 min to determine [CO2] in each chamber and 30 min to scan all the six chambers. The [CO2] thus determined was recorded and used to describe the CO2 regimes in the chambers. The rate of pure CO2 injection to maintain [CO2] in each chamber constant at the target level was controlled and measured by a mass flow controller (SEC-400MARK3, STEC, Kyoto, Japan), and recorded every 5 min for each chamber.
The net photosynthetic rate of the rice canopy on a ground area basis was determined as:
- ( Eqn 1)
(Pnet, canopy net photosynthesis rate (mg CO2 m−2 min−1); Cin, carbon injection rate to keep [CO2] in a chamber (mg CO2 m−2 min−1); L, chamber leakage rate (mg CO2 m−2 min−1); Rsoil, CO2 flux out of the paddy water and soil (mg CO2 m−2 min−1).) Canopy dark respiration rate on a ground area basis in night-time was determined as:
- ( Eqn 2)
(Rnight, canopy dark respiration rate in night-time (mg CO2 m−2 min−1); ΔC, increase of [CO2] in a chamber (mg CO2 m−2 min−1) while the air-ventilation is closed.) The air-ventilation system was programmed to allow for the measurement of ΔC, while maintaining the night-time [CO2] at desired level.
The rate of CO2 leakage (L) out of a chamber was estimated by modification of the method of Acock & Acock (1989) and Kimball (1990). Every few weeks pure N2O was injected into each chamber, and the decay of N2O concentration was measured by using the air sampling system described above and an infra-red gas analyser (ZRC1ZC11, Fuji-denki, Tokyo, Japan) for N2O. L was calculated from the measured leakage rate and the [CO2] gradient between ambient air and chambers by mass balance.
The CO2 flux out of the paddy water and soil was measured at air temperatures between 15 to 35°C with a 5°C step under flooded conditions, after all aboveground plant material had been removed at the end of the growing season.
The canopy dark respiration rate during the daytime (Rday) was calculated by assuming the same rate as that at night-time and at a corresponding temperature (Baker et al., 1997; Monje & Bugbee, 1998). Gross photosynthesis rate (Pgross) was estimated as Pnet plus Rday. The daily total respiration (Rtotal) was estimated as Rday plus Rnight and the daily carbon gain of a canopy (Cgain) was calculated as:
- ( Eqn 3)
The canopy net photosynthesis rate per unit leaf area (Pnet/Leaf area; Pleaf) and specific respiration rate (Rtotal/Total d. wt; Rdw) were calculated from destructive sampling date and a Pnet and Rtotal of few days before and after sampling.
Daily values of canopy photosynthesis, respiration and carbon gain were compared between the two [CO2] treatments for five periods: 11–40, 41–70, 71–100, 101–130, 131–153 DAT, and the entire growing season by one-way ANOVA with the variance between the chambers as the error variance.
Destructive sampling data, Pleaf and Rdw were also tested for significant effects of the [CO2] treatment by one-way ANOVA, but data of C and N analysis were tested by one-way ANOVA with the variance between the plants as error.
Graphing and smoothing of the daily measurement data were done with the computer software ‘Kaleida Graph’ (SYNERGY SOFTWARE, Reading, Pennsylvania, USA).