2.1 Plant material
Two Japanese cultivars of japonica rice, ‘Hinohikari’ and ‘Nikomaru,’ were used. Hinohikari rice is a common cultivar in warm temperate zones such as the Kyushu region in Japan, and Nikomaru rice is also cultivated in warm temperate areas and is a recently developed cultivar with higher heat tolerance. The seedlings were planted on June 13, 2018, in 1/5000 a Wagner’s pots (ø159 mm × 300 mm in height, approximately six L) filled with a flooded soil mixture of Andisol and Akadama soils (1:1) at three hills per pot and two seedlings per hill. Before planting, 1.013 g of N-P-K fertilizer (N-P-K = 15:15:15) (i.e., 76 kg N ha-1) and silica fertilizer (5.0 g) were applied to the pots. The seedlings were grown in six OTCs (60 cm in width, 120 cm in height, and 82.5 cm in length) located at Nagasaki University (Nagasaki, Japan) from June 26 to October 9. Inside each OTC, ambient air was introduced using a fan (MRS18V2-B, ORIENTAL MOTOR Co., Ltd., Japan) and was blown in an upward direction from the bottom of the chamber. For each cultivar, three pots were assigned to each chamber, and a total of six pots were placed on the floor of each chamber. The N-P-K fertilizer (1.013 g) was also applied on July 17 and August 25. Irrigation was conducted to keep the soil flooded during the cultivation period, except during drainage at the end of July. The air temperature (Tair) and relative air humidity (RH) both inside and outside of each chamber were continuously measured using a TR-72-wf Thermo Recorder (T&D Corporation, Nagano, Japan). The sensor of the recorder was set at a height of 115 cm from the bottom of each OTC, which corresponded to around the canopy height after rice heading. Each sensor was installed inside a ventilated two-layer radiation shield consisting of a fan (MU925S-11, ORIENTAL MOTOR Co., Ltd., Japan) and two polyvinyl chloride pipes with different diameters, the outer pipe being covered with an aluminum foil.
2.2 CO2 treatment
The rice plants were exposed to ambient or elevated CO2 concentrations in the OTCs from June 26 to October 9. Ambient air was introduced into three of the six OTCs assigned to the ambient CO2 treatment. In addition to ambient air, CO2 gas was introduced into the other three OTCs, assigned to the elevated CO2 treatment. To introduce CO2 gas, a polyethylene tube connected to a CO2 cylinder was inserted into the chamber near the outlet of the fan located at the lower part of the chamber. The target CO2 concentration in the elevated CO2 treatment was 550 ppm during the day, from before sunrise to after sunset. The introduction of CO2 gas was controlled manually by a valve with a flow meter, and the flow was stopped at night. The CO2 concentration inside the OTCs was monitored using a CO2 gas analyzer (LI-820, Li-Cor Inc., USA) and was continuously calibrated with standard CO2 gases (601 ppm and 374 ppm). To measure the CO2 concentration inside the chamber, the air inside each chamber at a height of 110 cm above the bottom was sampled sequentially using an electric valve system for a period of 5 min and introduced into the CO2 gas analyzer. The seasonal mean CO2 concentrations in ambient CO2 and elevated CO2 treatments during the day were 409.4 ± 0.6 ppm and 546.9 ± 3.1 ppm (mean of three chamber replications ± standard deviation), respectively. Although we did not measure the distribution of CO2 concentration inside the chamber throughout the experimental period, the range of the horizontal distribution at a height of 80 cm inside the chamber was approximately 95%–105% of the average. In each treatment, the pots were rotated within and among the chambers at 10–14-day intervals to minimize variation in chamber effects among the chambers.
2.3 Measurement of the leaf gas exchange rates
During the flowering period from August 22 to 27, 2018, the light-saturated net photosynthetic rate (A), stomatal conductance (gs), and transpiration rate (E) of the flag leaves were measured using an infrared gas analyzer system (LI-6400, Li-Cor Inc., USA). For each cultivar, three or four plants from each OTC were randomly selected for measurements. While the measurements were taken, air temperature, relative air humidity, and the photosynthetic photon flux density in the leaf chamber were maintained at 30 °C, 65%, and 1500 µmol m–2 s–1, respectively. For the measurements of A, gs, and E, the atmospheric CO2 concentration in the leaf chamber was 400 ppm for the ambient CO2 treatment and 550 ppm for the elevated CO2 treatment.
2.4 Measurements of the growth, yield, yield components, and grain appearance quality
To determine the heading date, we counted the stem and panicle numbers per plant and calculated the heading rate every day from August 21 to September 4. The heading date was defined as the day on which the mean heading rate reached 50% for each treatment. To measure the dry mass (DM) of plant organs, yield, and yield components, all rice plants of both Hinohikari and Nikomaru cultivars were harvested on October 7 and 9, 2018, respectively. The harvested plants were divided into panicles, leaf blades, stems (including leaf sheaths), and root parts. The separated plant organs, except for the panicle, were dried in an oven at 80 °C for 5 days and then weighed. The panicles were counted to obtain the panicle number per plant and then air-dried in the field for 5 days. Whole-plant DM was calculated as the sum of the DM of all plant organs. Grains were separated from dried panicles and counted to obtain the grain number per panicle. The grains were manually categorized into two groups, filled grains and unfilled grains, and counted. Filled grains were defined as fertile grains, including ripened and partially filled grains, and unfilled grains were defined as unfertilized grains. To evaluate spikelet fertility, the percentage of filled grains was calculated from the total grain number and the filled grain number for each plant. Filled grains were unhusked and weighed to obtain the yield per plant, and then the 1000-grain mass was calculated with the filled grain number per plant. The harvest index (HI) was the ratio of grain mass (yield) per plant to shoot (panicle, leaf blade, and stem) DM. Grain appearance quality was determined using a rice grain image analyzer (ES-1000, Shizuoka Seiki Co., Ltd., Japan), which classifies grains into perfect, immature, damaged, abortive, and colored grains (Sawada et al., 2016). Grain appearance quality was expressed as the percentage of the number of each quality class to the total grain number.
2.5 Statistical analysis
The mean of each parameter for each OTC was used for the statistical analyses (n = 3). Two-way analysis of variance (ANOVA) was used to test the effects of elevated CO2 treatment and cultivar. When there was a significant interaction between CO2 and the cultivar, Tukey’s HSD test was performed to identify significant differences among the four values. The HI, spikelet fertility, and grain quality were analyzed after logit transformation. All statistical analyses were performed using IBM SPSS Advanced Statistics 22.