Under P toxicity conditions, leaf withering would be caused by the oxidative stress resulting from the disruption of Cu/Zn-SOD activities as well as photosynthetic limitation. When PET activity exceeds the electron sink capacity, ROS production is stimulated in both PSII and PSI (Krieger-Liszkay, 2005; Sonoike, 2011; Pospíšil, 2016; Takagi et al., 2016a). ROS show high reactivity to biomolecules including DNA, proteins and lipids; therefore, the accumulation of ROS triggers cellular dysfunction to cause cell death (Apel & Hirt, 2004). Under high-Pi conditions, an increase was observed in the values of Y(NO) and Y(NA), which indicate the reduced states of PSII and PSI, respectively, despite an increase in the values of NPQ and Y(ND), which contribute to the suppression of ROS production both in PSII and PSI (Figure 2; Figures S1) (Müller et al., 2001; Takagi et al., 2017). Wada et al. (2018) reported that the Rubisco-antisense plants showed an over-reduction of the PET chain. Furthermore, Takagi et al. (2016b) reported that the suppression of electron sink activities inhibits both PET reaction andpmf generation. At the same time, the values of gH+ andV H+ also decreased because of the decrease in the ATP requirement of electron sink activities (Takagi et al., 2016b). Here, we observed that the ATP/ADP ratio significantly increased with increaseing Pi application (Figure 3d). Moreover,pmf , gH+, andV H+ decreased under 3.0 mM Pi conditions (Figure S2). Therefore, the high Pi accumulation would cause an over-reduction of the PET chain owing to a decrease in electron sink activities, including Rubisco deactivation. An increase in APX activity reconciles with the stimulation of oxidative stress under P toxicity conditions because the chloroplastic APX activity responds to the oxidative stress (Figure 5a, b) (Cakmak, 2005). Furthermore, a decrease in the Chl content which with the decreasing in PSI (Figure 1c; Figure S4), and the decrease in Fv/Fm supports the oxidative stress on the thylakoid membranes under excessive Pi application conditions (Figure 2j) (Terashima et al., 1994; Sonoike, 2011; Takagi et al., 2016a). In addition, we found an increase in the expression of an ROS-responsive gene PR5 (Ganesan & Thomas, 2001), and lipid hydroperoxide content (LOOH) under high-Pi application conditions (Figure S5a, c). In contrast, the expression of a programmed cell death-related gene VPE2 (Deng et al., 2011) decreased under high-Pi conditions (Figure S5b). Based on the illumination dependency of P toxicity symptoms (Delhaize & Randall, 1995), necrosis but not programmed cell death would be triggered by ROS production in thylakoid membranes, and this would be intensified by both the suppression of Cu/Zn-SOD activity and a decrease in photosynthetic electron sink capacities (Figure 9).
In the present study, we discussed a detailed mechanism for P toxicity in rice plants (Figure 9). To date, much attention has been paid to phytic acid synthesis in seeds. However, our results indicated that to maintain a proper leaf phytic acid content is important for maintaining plant growth and escaping oxidative stress triggered by PET reaction. Because the phytic acid synthesis pathway would be activated by an increase in the cytosolic Pi content through an increase in the sugar-phosphate content, the fine-tuning of Pi compartmentation within cells or modulation of sugar-phosphate metabolic flux might contribute to improving the Pi-use efficiency in the absence of phytic acid synthesis and P toxicity symptoms. The present study would open new opportunity to design breeding strategies for improving P use efficiency in crop plants. To modify the phytic acid content in land plants is also an important goal to improve human health because of severe Zn and Fe deficiency of humans in the world (Perera et al., 2018). That is, our findings would contribute to minimizing the phytic acid content as well as maximizing the plant yield for improving human health, especially for overcoming Zn-deficiency in humans and for fulfilling food demands in the future. Based on the present knowledge, we must continue to challenge ourselves to achieve these two important subjects in the main crop.