DISCUSSION
Owing to the low concentrations of Pi in the soil (< 10 µM) (Bieleski, 1973), Pi acquisition often limits plant growth and crop yield. For this reason, improving Pi absorption and utilization in land plants is one of the molecular targets for increasing agricultural productivity (Gu et al., 2016). On the other hand, excessive Pi absorption in plants causes P toxicity (Gu et al., 2016). Therefore, to improve Pi-use efficiency in crops, P toxicity must be avoided, and the mechanisms of P toxicity should be understood. However, the detailed mechanisms of illumination-dependent P toxicity that causes leaf withering in land plants remain to be clarified (Cakmak & Marscher, 1987; Delhaize & Randall, 1995; Ova et al., 2015). To address this question, we characterized the effects of different Pi application conditions on rice plants from the aspect of photosynthesis. Here, we propose three important factors underlying P toxicity symptoms: activation of the phytic acid synthesis pathway in the leaves, limitation of photosynthesis, and disruption of Cu/Zn-SOD activity.
We suggest that P toxicity is triggered by activating phytic-acid synthesis and decreasing metal availability in leaves. In seeds, phytic acid synthesis is stimulated in response to Pi accumulation for storing P and metals (Hawkesford et al., 2012; Su et al., 2018). In line with this observation, we found that phytic acid synthesis substantially occurs with an increase in Pi accumulation in the leaves due to the upregulation of the gene expressions involved in phytic acid synthesis (Table 1; Figure 8). The activation of phytic acid synthesis would be triggered by an increase in G6P content in the cytosol (Figure. 3b). Under higher Pi application conditions, the sugar-phosphate content increased in both chloroplasts and cytosols (Figure. 3b). The triose-phosphate translocator (TPT) exports 3-PGA and triose-phosphate between chloroplasts and cytosol by exchanging Pi (Flügge & Heldt, 1984). An increase in the cytosolic Pi level would stimulate the export of triose-phosphate from the chloroplasts to the cytosol (Flügge, 1992), and the metabolic flux involving sugar-phosphates (Figure 3a, b). The phytic-acid synthesis occurs by two distinct pathways, lipid-dependent and lipid-independent (Figure 8a) (Suzuki et al., 2007; Perera et al., 2018). The lipid-independent pathway responded to an increase in Pi accumulation in the leaves, and an increase in the phytic acid/free Pi ratio suggests that certain proportion of Pi is converted to phytic acid in accordance with the sugar-phosphate accumulation (Figure 3b; Figure 8b-e). Phytic acid has high metal-chelating activity, and Maenz et al. (1999) showed that Zn, rather than other metals, is an exclusive target of phytic acid. Thus, an increase in ZIP4 expression suggests that phytic acid captures Zn within cells and insolubilizes it (Figure 7b). In fact, Cakmak and Marschner (1987) observed that Pi accumulation increased the insoluble Zn content in the leaves. Furthermore, the cytosolic pH (around 7.5) is optimal for phytic acid to chelate Zn (Maenz et al., 1999; Mimura et al., 2000). Previous studies showed that phytic acid is synthesized in the cytosol, and is stored in the vacuoles of the leaf cells (Mitsuhashi et al., 2005; Nagy et al., 2009; Lee et al., 2015). Moreover, the suppression of the Pi transport to vacuoles stimulates the P toxicity symptoms (Liu et al., 2015; Liu et al., 2016). Based on these studies, an increase in the cytosolic Pi accelerates triose phosphate export and G6P synthesis, thereby stimulating the synthesis of phytic acid, which, in turn, insolubilizes Zn in the cytosol. This can explain the previous observation of P toxicity symptoms similar to Zn deficiency symptoms in land plants, despite substantial accumulation of Zn in land plants (Cakmak & Marscher, 1987; Ova et al., 2015). In contrast to ZIP4 , COPT1 expression showed no significant response to an increase in Pi accumulation in the leaves, implying that a decrease in Cu/Zn-SOD activity was caused by the suppression of Zn availability in cells. Fe is also a target of phytic acid accumulation. In fact, IRO2 expression increased with increasing Pi accumulation (Figure 7b) (Maenz et al., 1999). However, a decrease in Fe- and Mn-SOD activities was not found (Figure 5c). APX also contains Fe as a co-factor, and its activity decreased under Fe-deficiency conditions (Ranieri et al., 2001). However, the APX activity was rather increased under high-Pi conditions (Figure 5a, b and Figure S3b, c). These results suggest that Fe-deficiency is not severe than Zn-deficiency within cells under P toxicity conditions. Interestingly, IMP1-1 expression was activated under low-Pi conditions (Figure 8b). The phytic acid/free Pi ratio showed a response similar to that of IMP1-1 expression (Figure 8e). These results suggest that the lipid-dependent phytic acid synthesis pathway could be activated under low-Pi conditions. Although phytic acid is a major storage-form of Pi in seeds (Hawkesford et al., 2012), the inhibition of phytic acid synthesis led to leaf morphology distortion inArabidopsis or growth retardation in crop plants (Raboy et al., 2000; Stevenson-Paulik et al., 2005). Lee et al. (2015) demonstrated that phytic acid is required for activating LOS4/Gle1-mediated mRNA export from nucleus in Arabidopsis in a manner similar to animals and yeast, and the phytic acid deficiency causes mRNA accumulation within nuclei. In addition, phytic acid inactivates inward K+ conductance by mobilizing Ca2+ in the plasma membranes in response to abscisic acid in the guard cells and contributes to pathogen defense (Lemtiri-Chlieh et al., 2000, 2003; Murphy et al., 2008; Nagy et al., 2009). Based on these studies, the lipid-dependent pathway might secure phytic acid synthesis under low-Pi availability conditions to maintain the phytic acid dependent physiological reactions. Further investigation is required to elucidate the validity of this hypothesis.
The limitation of photosynthesis would be enhanced by a decrease in the RCA content. We found that Pi accumulation decreases electron sink capacities by deactivating Rubisco, but not lowering Rubisco content (Figures 2 and 4). Furthermore, the α - and β -forms of RCA were significantly decreased with an increase in Pi application (Figure 4e-i). Among the three RCA iso-forms, the α - and β -forms are the major components of Rubisco activation (Zhang et al., 2002; Portis, 2003), and it has not been addressed whether the β* -form has the ability to activate Rubisco. These results suggest that the target of P toxicity exists not in Rubisco itself, but within the isoforms of RCA, especially α - and β -forms. In the RCA mutants of Arabidopsis and rice plants, A linearly increases with increasing Ci in a manner similar to our results under high Pi conditions in WT plants (Zhang et al., 2002; Masumoto et al., 2012; Yamori et al., 2012). Although we could not elucidate the detailed mechanism to differentiate the content of RCA isoforms in the leaves, Vargas-Suárez et al. (2004) suggested that the β* -form is generated by limited proteolysis under stress conditions, such as drought, and changing the β- form to β*- form resulted in higher chaperone activity. From this point, we suggest the possibility that the β* -form is upregulated to protect proteins under the oxidative stress conditions caused by P toxicity at the cost of Rubisco activation. Supporting this idea, Rokka et al. (2001) suggested that RCA plays a second role as a chaperone for protecting thylakoid membrane proteins under heat stress conditions, that is, the physiological functions of RCA would not be limited to activate Rubisco.
We suggest that the severe limitation of photosynthesis is caused by additional factors in concert with a decrease in the RCA content. Badger & Lorimer (1981) reported that Pi acts as a weak Rubisco inhibitorin vitro , that is, Rubisco activation might be further declined under P toxicity conditions in vivo . Moreover, Suzuki et al. (2012) reported that when Rubisco severely limits the photosynthetic metabolic flux in the leaves, the RuBP content is increased. Here, we observed that an increase in the sugar-phosphate content of the latter part of GA3P and DHAP rather than an increase in RuBP (Figure 3b). This result suggested that another target, besides RCA, should be suppressed under P toxicity conditions.