*4.1. Plant Material and Growing Condition*

The low-phytate soybean line CX1834, isolated from the mutant originally referred to as M153 [14], was obtained from USDA-ARS, National Small Seeds Germplasm Research Unit, Aberdeen, Idaho. This low-phytate line was used as the pollen parent in a cross with the Japanese commercial cultivar "Tanbakuro" [21]. These two parents were crossed in August 2004 under greenhouse conditions, and ten progeny populations were developed from the F1 seeds. The F1 generation and the parents were grown in a field of the Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan, during the summer of 2005, and low-phytate lines were selected by measuring the phytate and inorganic and total phosphorus concentrations in seeds after harvesting. In this study, we conducted two experiments using one selected low-phytate line each (F10 line for Experiment 1 and F11 line

for Experiment 2) and one normal-phytate cultivar each (Enrei for Experiment 1 and Akimaro for Experiment 2), that are cultivated in western Japan. Both the low-phytate and normal-phytate soybeans were sown in a seed bed containing a soil mixture of granite regosol soil, perlite, peat moss, and nursery soil with compost (2:1:1:1 per volume base). Inoculation of root nodule bacteria (*Bradyrhizobium japonicum*) was performed by mixing a small amount of soil from the previous year's cultivation into the seedbed. Both experiments were conducted in the greenhouse for prevention of rainfall.

Experiment 1: Both soybean F10 line and cv. Enrei were planted in seed beds containing a mixture of soil as above on 3 June, 2016. After 13 days of germination, uniform seedlings were transplanted to a wooden container (30 cm in width, 10.5 m in length, and 18 cm in depth) filled with a mixture of soil. The plant-to-plant space was 20 cm in containers separated by 100 cm distance from each other. Each treatment was separated by inserting a water proof plastic sheet in order to prevent moisture and nutrient movement between the treatments in the container. Before transplanting, the soil mixture was fertilized with a basal fertilization of 100 kg ha−<sup>1</sup> of K2O as potassium sulfate. The soil pH (H2O) was adjusted to about 6.0 with dolomitic calcium carbonate. P treatment provided the following two fertilization levels: 50 and 150 kg P2O5 ha−<sup>1</sup> as single super phosphate in a randomized complete block design with four replications. At the flowering stage in mid-July, the leaf photosynthetic rate and nitrogen fixation of the nodule were measured. At the full-maturity stage in mid-October, plants were harvested and the stem was separated. The seeds and stem were dried at 80 °C in an air-forced oven for more than 3 days, and weighed.

Experiment 2: Both soybean F11 line and cv. Akimaro were planted in seed beds containing a mixture of soil on 11 June, 2017. After 14 days of germination, one seedling each was transplanted to 9 liters pots (25 cm in diameter, and 23 cm in depth) filled with soil mixture. The pot-to-pot distance was 30 cm with no interaction between the plant roots. Before transplanting, as in Experiment 1, the soil mixture was fertilized with a basal fertilization of 100 kg ha−<sup>1</sup> of K2O as potassium sulfate. The soil pH (H2O) was adjusted to about 6.0 with dolomitic calcium carbonate. P treatment provided the following two fertilization levels: 20 and 100 kg P2O5 ha−<sup>1</sup> as single super phosphate in a randomized complete block design with four replications. The plants were harvested at full-maturity stage in mid-October. After pod and seed numbers were counted and the seeds were weighed, the seeds were oven dried at 70 °C for 72 h for analysis of the mineral and oil concentration.

### *4.2. Determination of Leaf Photosynthesis and Dinitrogen Fixation*

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<sup>−</sup><sup>2</sup> <sup>S</sup>−1, the humidity was 65%, the leaf temperature was 28 °C, 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.
