Gc 14b

The dinitrogen fixation activity and leaf photosynthetic rate were determined at flowering stage in Experiment 1. The photosynthetic rate was measured on the upper most fully-expanded leaf using portable infrared gas analyzer (Model LI-6400, Licor Co. Ltd., NE, USA) between 10:30 a.m. and 12:30 p.m. While taking the measurements, the photosynthetic active radiation was adjusted at 1200 µmol m⁻² S⁻¹, the humidity was 65%, the leaf temperature was 28 ℃, and the ambient CO₂ concentration was 350 mol L⁻¹. Dinitrogen fixation was analyzed by measuring the reduction of acetylene to ethylene at flowering time as follows. Intact root systems were excised and gently separated from the soil. The root system and attached nodules were quickly placed in a 1000 mL glass bottle. The bottle was sealed with a rubber stopper and 100 mL of air in the bottle was replaced with acetylene. Samples were incubated at ambient laboratory temperatures. During the incubation periods, 0.3 mL of gas samples were extracted at 10 min and 1 hour and injected into a gas chromatograph (Shimazu GC-14B, Shimazu Co., Kyoto, Japan) fitted with a flame ionization detector to determine the ethylene concentration. Specific nitrogenase activity was calculated by dividing the nitrogenase activity of each sample by the dry weight of the nodules.

The emission of CH₄ was measured weekly or biweekly between 7 June and 23 August using a closed chamber method, as described previously (16 ). Each chamber consisting of lower (60 cm H) and upper (60 cm H) sections was placed over 4 hills of rice plants with a basal area of 30 × 60 cm. The upper section of the chamber fit over the lower one and was supported by a water-filled groove surrounding the outer top lip of the lower section, thereby providing an airtight seal between the two sections and surrounding atmosphere. Gas samples were collected from the chamber 0, 10, and 20 min after placement of the chamber. The samples were injected into pre-evacuated 19-mL glass vials and brought back to the laboratory for analysis. The mixing ratio of CH₄ was determined by gas chromatography equipped with a flame ionization detector (GC-14B; Shimazu, Kyoto, Japan). The emission of CH₄ was calculated based on an increase in the mixing ratio of the basal area of the chamber, chamber volume, and temperature inside the chamber. An analysis of variance (ANOVA) was conducted on the cumulative amount of CH₄ that was emitted during 41–90 days after transplanting (DAT) using a general linear procedure. [CO₂], temperature, and [CO₂]×temperature were treated as fixed effects, while ring and ring×[CO₂] were treated as random effects.

We set an approximately 2 m long and 0.5 m wide ladders from the center of the shorter bund to allow measurement of CH
₄ without touching the paddy soil surface. Those ladders were on the borders of the non-planted areas in each field. We set PVC chamber bases on the paddy fields of both sides of the ladders to avoid measurement perturbation. Chambers (60 × 80 cm and 100 cm high, transparent acryl) were set on a watertight chamber bases for every measurement. Measurements were taken at 8 a.m. because previous research has indicated that emissions at this time have a high correlation (ca. 90% of average emission) with average daily emissions
²² . We mixed the air in the chamber with a fan for 5 min after setting the chamber, then sampled the first gas, then sampled the second gas 20 min later. We conducted the measurements once a week throughout the rice growing stage, but every 3 days for 2 weeks after seeding, heading stage, and around draining. The samples were analyzed by gas chromatography (GC-14B, Shimazu, Kyoto). The cumulative CH
₄ emissions were calculated by linear interpolation.