*4.4. Analysis of xylem sap*

Rice was grown for 4 weeks hydroponically in a growth chamber as described by Nagai et al. [38]. The shoots were cut 3–4 cm above the shoot meristem; the section was covered with silicon tube containing glass wool, and then xylem sap was collected for 2 h (10:00–12:00 h). The glass wool was transferred into an ultrafiltration filter tube (Nanosep, ODGHPC34; Pall Corporation, NY, USA) and centrifuged at 17 800 g for 3 min. Xylem saps were stored at –80 ◦C. Xylem sap diluted 10 times with distilled water was used to determine the Pi content. Diluted saps (50 μL) were treated with 25 μL of color reagen<sup>t</sup> containing 10% (w/v) ascorbic acid, 97% (w/w) H2SO4, and 5% (w/v) ammonium molybdate. After incubation at 40 ◦C for 1 h, the absorbance of the sample at 655 nm was measured using a microplate spectrophotometer (xMark, Bio-Rad, Hercules, CA, USA). A P calibration curve was prepared using P standard solution.
