Characterization of the rice PHO1 gene family reveals a key role for OsPHO1;2 in phosphate homeostasis and the evolution of a distinct clade in dicotyledons

Abstract

Phosphate homeostasis was studied in a monocotyledonous model plant through the characterization of the PHO1 gene family in rice (Oryza sativa). Bioinformatics and phylogenetic analysis showed that the rice genome has three PHO1 homologs, which cluster with the Arabidopsis (Arabidopsis thaliana) AtPHO1 and AtPHO1;H1, the only two genes known to be involved in root-to-shoot transfer of phosphate. In contrast to the Arabidopsis PHO1 gene family, all three rice PHO1 genes have a cis-natural antisense transcript located at the 5 ' end of the genes. Strand-specific quantitative reverse transcription-PCR analyses revealed distinct patterns of expression for sense and antisense transcripts for all three genes, both at the level of tissue expression and in response to nutrient stress. The most abundantly expressed gene was OsPHO1;2 in the roots, for both sense and antisense transcripts. However, while the OsPHO1;2 sense transcript was relatively stable under various nutrient deficiencies, the antisense transcript was highly induced by inorganic phosphate (Pi) deficiency. Characterization of Ospho1;1 and Ospho1;2 insertion mutants revealed that only Ospho1;2 mutants had defects in Pi homeostasis, namely strong reduction in Pi transfer from root to shoot, which was accompanied by low-shoot and high-root Pi. Our data identify OsPHO1;2 as playing a key role in the transfer of Pi from roots to shoots in rice, and indicate that this gene could be regulated by its cis-natural antisense transcripts. Furthermore, phylogenetic analysis of PHO1 homologs in monocotyledons and dicotyledons revealed the emergence of a distinct clade of PHO1 genes in dicotyledons, which include members having roles other than long-distance Pi transport.

Figure 1.

Phylogenetic analysis of the PHO1 family in monocotyledons and dicotyledons. A, Unrooted phylogenetic tree of the PHO1 family from rice (Os), sorghum (Sb), maize (Zm), B. distachyon (Bd), Arabidopsis (At), papaya (Cp), poplar (Pt), grapevine (Vv), and barrel medic (Mt). The tree was made using the neighbor-joining method. The two major clades are encircled, the Arabidopsis genes are underlined, and the rice genes are in bold. B, Identity/similarity matrix for rice and Arabidopsis PHO1 proteins. Amino acid identity and similarity are indicated by first and second number, respectively.

Figure 2.

Structure of the OsPHO1 genes with their respective cis-NAT. Exons are indicated by black boxes. Numbers indicate the length of the mRNA, in base pairs, and the large arrows indicate the orientation of transcription. Small arrows indicate the location of the primers used for strand-specific qRT-PCR.

Figure 3.

Spatial expression of the OsPHO1 transcripts. Sense and antisense transcript levels for OsPHO1;1 (A), OsPHO1;2 (B), and OsPHO1;3 (C) in 7-d-old seedlings, flowers before, during, and after pollination, leaf sheath, leaf blade, and roots are represented. Apart from seedlings, all other tissues were harvested from plants grown in soil for 5 to 6 months in a greenhouse. Plants were fertilized weekly with a nutrient solution containing 1 mm Pi. All data are means of three replicates with error bars indicating sd, and represented as transcript level per 10⁶ copies of ubiquitin.

Figure 4.

Expression pattern of the OsPHO1 transcripts in response to different nutrient deficiencies. After a week of pregermination in water, plants were grown for 2 weeks in complete medium (Ctrl) or in medium deficient in sulfur (-S), phosphate (-Pi), nitrogen (-N), potassium (-K), or iron (-Fe), before being harvested. Expression levels of both sense (A, C, and E) and antisense (B, D, and F) transcripts were assessed using strand-specific qRT-PCR for OsPHO1;1 (A and B), OsPHO1;2 (C and D), and OsPHO1;3 (E and F). All data are means of three replicates with error bars indicating sd, and represented as transcript level per 10⁶ copies of ubiquitin.

Figure 5.

Expression pattern of the OsPHO1 transcripts in response to Pi starvation. Strand-specific qRT-PCR was performed to detect the levels of expression of sense (white bars) and antisense (black bars) transcripts of OsPHO1;1 (A and B), OsPHO1;2 (C and D), and OsPHO1;3 (E and F) transcripts in shoots (A, C, and E) and roots (B, D, and F). After a week of pregermination in water, the plants were grown for a week in presence of 1 mm Pi before being transferred to Pi-deficient media (T = 0 d) for the desired period of time (from 0–30 d). After 30 d of Pi starvation, plants were transferred to Pi-sufficient conditions for 1 to 4 d. Control plants (+Pi) were grown for 34 d in presence of 1 mm Pi. All data are means of three replicates with error bars indicating sd, and represented as transcript level per 10⁶ copies of ubiquitin. Pi content in the aerial part (G) and in the roots (H) is represented. Error bars are sd, n = 6.

Figure 6.

Functional characterization of Ospho1;1 mutants. A, Positions of Tos17 and T-DNA insertions in the OsPHO1;1 gene are indicated by arrowheads. Black boxes represent exons and black lines represent introns. Small arrows represent the gene-specific primers (FL1 and RL1) used for RT-PCR. B, RT-PCR using RNA of roots from mutants and wild-type (WT) plants. C, Morphological appearance of Ospho1;1 null mutants plants compared to the wild type. Plants were pregerminated in water for 7 d and then grown hydroponically for 6 weeks in presence of full Hoagland medium (1 mm Pi), before being harvested. Black bar represents 10 cm. D and E, Fresh weight (FW; D) and Pi content (E) were assessed for shoots and roots of Ospho1;1 mutants and the wild type after 6 weeks of growth in medium with 1 mm Pi. All data are means of six replicates with error bars indicating sd. [See online article for color version of this figure.]

Figure 7.

Functional characterization of rice Ospho1;2 mutants. A, Positions of Tos17 insertions in the OsPHO1;2 gene are indicated by arrowheads. Black boxes represent exons and black lines represent introns. Small arrows represent the gene-specific primers (FL2 and RL2) used for RT-PCR. B, RT-PCR using RNA of roots from mutants and wild-type (WT) plants. C, Morphological appearance of Ospho1;2 null mutants plants compared to the wild type. Plants were pregerminated in water for 7 d and then grown hydroponically for 4 weeks in presence of full Hoagland medium (1 mm Pi), before being harvested. Black bar represent 10 cm. D, Fresh weight was assessed for shoots and roots of Ospho1;2 mutants and the wild type after 4 weeks of growth in medium with 1 mm Pi. E, Pi content was assessed in roots and shoots for Ospho1;2 mutants and the wild type. Plants initially germinated in water for 7 d and grown for an additional 7 d in medium with 1 mm Pi (time 0) before transferring them to medium without Pi for 10, 20, and 30 d. After 30 d in Pi-deficient medium, plants were transferred back in medium with 1 mm Pi for 2 d (30 + 2 d). Control plants (right histograms +Pi) were grown for 34 d in medium with 1 mm Pi. FW, Fresh weight. F, Translocation rate of Pi from roots to shoots of 3-week-old plants grown hydroponically in Pi-deficient medium. All data are means of six replicates with error bars indicating sd. [See online article for color version of this figure.]

Figure 8.

Expression of the OsPHO1 gene family in the Ospho1;2 mutants. Strand-specific qRT-PCR was performed to detect the levels of expression of sense (white bars) and/or antisense (black bars) transcripts of OsPHO1;1 (A and B), OsPHO1;2 (C and D), and OsPHO1;3 (E and F) transcripts in shoots (A, C, and E) and roots (B, D, and F) in the wild type (WT) and in the two Ospho1;2 mutants. After a week of pregermination in water, the plants were grown for 5 weeks in medium containing 1 mm Pi, before harvesting shoots and roots separately. All data are means of three replicates with error bars indicating sd, and represented as transcript level per 10⁶ copies of ubiquitin.