SN9816, OsATG8b-overexpressing lines, and osatg8b mutants were grown hydroponically with NS or NL nutrient solution as above. The 15N-Labeling experiment was conducted as previous studies described [5, 14] at the booting stage (70 days after sowing) when most of the 11th leaves on the main stem just emerged from the sheath of the 10th leaves, the nutrient solution was replaced with the 15N-labeled (15NH4)2SO4 and Ca(15NO3)2·4H2O (10.6 atom% excess) as 15NS or 15NL nutrient solution for 3 d. Afterward, the pots were applied with NS or NL solution every 3 days until plant maturity, then plants were separated into seed, stem, leaf sheath, and leaf blade. The 15N abundance, 15NHI, and NRE were estimated as described [20]. All the above traits were measured on a single-plant basis, and 12 plants under each N conditions were randomly selected as replicates to measure.

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