*2.4. PA-P and Pi Content*

The Pi content and PA-P/Pi ratio at 10 days after flowering (DAF) and at harvest are shown in Figure 3. We found that the Pi content showed no response to P fertilizer treatment (Figure 3a). WRC 6 showed a significantly higher Pi content at 10 DAF than WRC 5. The PA-P/Pi ratio presented no significant difference between P treatments or genotypes at 10 DAF (Figure 3b). The PA-P/Pi ratio at harvest was increased under P4 treatment and was associated with the PA content per grain (Figures 2b and 3b).

**Figure 3.** Effect of the amount of P fertilizer on Pi and PA-P/Pi in WRC 5 and WRC 6. **(a**) Pi content per gram dry weight (μg/grain) and (**b**) PA-P/Pi ratio at 10 DAF and harvest. Gray and blue represent the value at 10 DAF and harvest, respectively. Each value represents the mean ± SD of four replicates. Different letters indicate statistical differences (Tukey's HSD test, *p* < 0.05).

### *2.5. INO1 Expression and PA Content at 10 DAF*

The expression of *INO1* at 10 DAF was significantly increased by P fertilizer treatment in WRC 6, whereas WRC 5 showed no response to P fertilizer treatment (Figure 4). We also investigated the expression levels of eight PA biosynthesis genes in Nipponbare (Figure 5). Among these eight genes, no significant differences in the expression level were observed except for the multidrug resistance-associated protein 13 (*MRP13*) gene, which showed a significant decrease under the P4 treatment.

**Figure 4.** Relative gene expression of *INO1* at 10 DAF in WRC 5 and WRC 6. White and pink indicate the control and P treatment, respectively. Each value represents the mean ± SD of three replicates. Different letters indicate statistical differences (Tukey's HSD test, *p* < 0.05).

**Figure 5.** Expression level of PA biosynthesis genes in Nipponbare. White, pale pink, and pink indicate the control, P1 treatment, and P4 treatment, respectively. Each value represents the mean ± SD of three replicates. Different letters indicate statistical differences (Tukey's HSD test, *p* < 0.05).

### *2.6. Short-Term Response of Pi Uptake in WRC 5 and WRC 6*

To evaluate Pi uptake on WRC 5 and WRC 6 in the initial growth stage, we measured the Pi content in xylem sap using the molybdenum blue method (Figure 6). When additional P fertilizer was applied, the Pi content in xylem sap per root dry weight (μg/gDW/<sup>2</sup> h) was significantly increased, whereas there was no significant difference between WRC 5 and WRC 6.

**Figure 6.** Short-term response of Pi uptake using the xylem sap analysis in WRC 5 and WRC 6. (**a**) Pi content in xylem sap oozed out from one seedling for2h(μg/plant/<sup>2</sup> h) and (**b**) Pi content per root DW (μg/gDW/<sup>2</sup> h). White and pink indicate the control and P treatment, respectively. Each value represents the mean ± SD of three replicates. Different letters indicate statistical differences (Tukey's HSD test, *p* < 0.05).
