**1. Introduction**

Many people living in developing countries have a higher risk of malnutrition due to Zn deficiency, as they mainly take micronutrients from cereals, such as legumes, wheat, and rice. Atmospheric carbon dioxide (CO2) concentration is increasing and expected to reach 700 ppm by the end of this century [1]. Elevated CO2 (e[CO2]) causes a reduction in the mineral content in staple crops [2]. In the future, Zn deficiency is expected to expand globally, especially in developing countries, due to e[CO2]. Phytic acid (*myo*-insitol-1,2,3,4,5,6-hexakisphate; PA) is the storage form of P in cereal grains; it accounts for 75% of total P in grains [3]. Phytic acid is also known to chelate with minerals, such as Fe, Mg, Ca, K, and Zn. It prevents the absorption of minerals in the human intestine and is regarded as an antinutrient. Research conducted using a suckling rat pup model showed a negative correlation between dietary PA and Zn absorption from the grains of maize, rice, and barley [4]. In addition, PA inhibits enzymes needed for protein degradation and disturbs proteolysis in the stomach and small intestine [5]. Ruminants, such as cows, secrete phytase, an enzyme digesting PA, but humans lack phytase and therefore cannot digest PA. Thus, reducing the PA content in cereals is essential to overcome Zn deficiency.

The regulation of PA content has to take into account the P status in plants, P absorption from the roots, and remobilization from plant organs. P transporters are known to be involved in the uptake of inorganic P (Pi) from the root and transport to plant organs. So far, 13 Pi transporters belonging to the PHT1 family have been identified in rice [6,7]. OsPT8 known as a high a ffinity Pi transporter is essential for Pi translocation from vegetative organs into rice gain [8]. SULTR-like phosphorus

distribution transporter (SPDT) controls the allocation of P to the grains [9]. These findings sugges<sup>t</sup> that it might be possible to control the PA content by manipulating P transporter.

To identify the biosynthetic pathway of PA and reduce the PA content in the grain, low phytic acid (*lpa*) mutants of wheat [10], maize [11–13], soybean [14,15], barley [16–18], and rice [19] have been used. These mutants have disrupted PA biosynthesis genes and exhibit low PA accumulation in the grains; however, in most cases, these mutants showed a significant reduction in germination and yield [20]. On the contrary, mutants that repress *INO1* with an 18-kDa oleosin promoter showed an approximately 70% reduction in the PA content, with no negative e ffects on plant growth [21]. Similarly, no undesirable yield reduction was observed in *lpa* mutants of barley [22] and soybean [23]. Developing stable *lpa* mutants without yield loss is indispensable to overcome malnutrition.

P is one of the most essential elements for plant growth. P fertilizer is indispensable in practical agriculture and therefore P application cannot be reduced [24]. The applied P is absorbed from the root and remobilized to the shoot, and then PA is synthesized in developing seeds using the transported P [25]. It has been reported that the PA content is a ffected by the amount of supplied P in various crops [26–28], including rice [29]. In a low-phytate soybean line, which derived from a cross of the normal-phytate Japanese cv. Tanbakuro and the low-phytate line CX1834, no negative e ffects were reported on plant growth and yield, leaf photosynthesis, and nitrogen fixation by di fferent levels of P fertilizer application [30]. These results sugges<sup>t</sup> that the *lpa* mutant can exhibit a low PA phenotype without any loss of growth-related performances at various levels of P fertilizer application. Thus, information on the e ffect of P fertilizer on seed PA content and yield in the *lpa* mutant is accumulating; however, it is not clear how P fertilizer a ffects the PA content in natural genotypic variants.

Previously, we examined variations in the PA content in 69 accessions of the World Rice Core Collection (WRC) and identified WRC 5 and WRC 6 as cultivars with the lowest and highest PA content in the collection [31]. We then compared WRC 5 and WRC 6 to identify the molecular determinant of the natural variation in the PA content in rice. The results suggested the gene expression level of *myo*-inositol 3-phosphate synthase 1 (*INO1*) was the genetic basis explaining the natural variation in PA accumulation in rice [32]. Interestingly, DNA sequences of the coding and promoter region (1000 bp) of the *INO1* gene were identical between WRC 5 and WRC 6. This suggests there are di fferent regulation mechanisms of PA content besides DNA mutation of the biosynthesis gene. To elucidate these regulation mechanisms, WRC 5 and WRC 6 will be useful cultivars. In this study, we evaluated the e ffect of P fertilization on the PA content in WRC 5 and WRC 6 to clarify how the PA content is regulated under a di fferent P status in natural variants of rice.
