**4. OsPht1;4 Phosphate Transporter**

The OsPht1;4 (or OsPT4, corresponding to LOC\_Os04g10750) phosphate transporter, belonging to the Pht1 family, was described as influencing grain PA content, as the corresponding mutant produces *lpa* grains [21]. As the identification of putative orthologs of this protein in other species is not so obvious, due to the high number of Pht1 genes and to their sequence similarity, (in rice there are 26 [97]), in the present review we limit our consideration to OsPT4.

The genomic sequence is characterized by the presence of a single exon (Figure 5a) and the protein, 538 aa long, by a major facilitator superfamily domain, characteristic of di fferent transporters, including phosphate transporters (Figure 5b).

**Figure 5.** (**a**) Gene structure of OsPHT1;4. Light and dark blue rectangles represent UTRs and coding exons, respectively, the black bars correspond to introns. The gene structure was obtained as described in Figure 1a legend. (**b**) Predicted domain of the OsPHT1;4 protein by PFAM [98] software. The major facilitator superfamily (MFS) domain is represented.

The *OsPT4* gene is mainly expressed in roots, flag leaves and embryos, and its expression is increased in response to prolonged P starvation conditions in shoots and roots, where the signal is specifically localized to the exodermis. The protein is localized to the plasma membrane, as shown in the protoplast system and it is a functional Pi influx transporter, able to complement a yeas<sup>t</sup> mutant defective in Pi uptake and to facilitate the increased accumulation of Pi in Xenopus oocytes.

### *Ospt4 Mutants, OsPT4 RNAi and Overexpression Lines*

The OsPT4 functional characterization was performed using transposon insertional mutants, knockdown lines harboring the *OsPT4-RNAi* construct and overexpression lines. Pi and total P concentration is strongly reduced in mutant lines, attenuated in RNAi lines and increased in overexpression lines, in roots as well as in shoots. Moreover, a dramatic reduction in Pi uptake in mutants, a small reduction in RNAi lines, and an increase in overexpression lines were observed [21,22]. There was altered expression of genes that regulate Pi absorption and homeostasis, such as *OsPHO2*, *OsPHR2*, and *OsSPX1*. A detailed analysis of the grains revealed a decrease in total P concentration in the embryo and in one line also in the endosperm, attenuated and increased e ffects in RNAi lines and overexpression lines, respectively. Moreover, a decrease of 32–22% in PA concentration was observed in *ospt4-1* and RNAi lines' grains and an increase of 10% in overexpression lines. The alteration in mutant and RNAi embryos correlates with a reduction in the transcript levels of *OsRINO* (coding for *myo*-inositol 3-phosphate synthase—MIPS) and of *OsIPK1* (coding for 1,3,5,6-pentakisphosphate 2-kinase), coding for enzymes catalyzing the first and the last step of the PA biosynthetic pathway, respectively. Both genes' transcript levels were also significantly reduced in the endosperm of mutant grains and only partially reduced in RNAi lines [21]. From all these data, it is clear that OsPT4 has an important role in acquisition and mobilization of Pi and also during embryogenesis and seed development, so it is a good candidate to improve P e fficiency, although alterations in panicle robustness, grain-setting rates, grain weight, grain yield per plant and seed germination registered in *ospt4* and *OsPt4* RNAi lines need breeding actions to ensure acceptable agronomic performance and avoid yield penalties.
