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.