Fig. 1. Phylogenetic tree analysis of PHT4 family members.Maximum-likelihood phylogenetic analysis using PHT4 protein sequences from 12 different plant species and 1 chlorophyta. The different colours indicate different PHT4 families: blue, PHT4;1; cyan, PHT4;2; yellow, PHT4;3; purple, PHT4;4; green, PHT4;5; pink, PHT4;6; and black, Other clustering trees. Letters in the codes represent species names as follows: At, Arabidopsis thaliana; ATR, Amborella trichopoda; Cre, Chlamydomonas reinhardtii; Cucsa, Cucumis sativus; Mapoly, Marchantia polymorpha; Os, Oryza sativa; PAB, Picea abies; Potri, Populus trichocarpa; Pp, Physcomitrella patens; SMO, Selaginella moellendorffii; Solyc, Solanum lycopersicum; UGI.scf, Utricularia gibba; Zm, Zea mays.

Supplemental Fig. 1. Transcript profiles of PHT4 family members in spatial temporal expression data in RiceXPro (https://ricexpro.dna.affrc.go.jp/GGEP/).

Fig. 2. Transcript profiles of PHT4 family members in various organs in rice. The expression levels of OsPHT4s were represented as relative to the respective value of shoots at the vegetative stage. VS, Vegetative stage (21-day-old plants); FS, Flowering stage (48-day-old plants); GFS, Grain filling stage (60-day-old plants). Leaf 1?7 refer to true leaves from the 1st to the 7th.Data are Mean ± SD (n = 3). *, P ≤ 0.05 and **, P ≤ 0.01 by the Student’s t-test.

Fig. 3. Subcellular localization of OsPHT4 genes in rice protoplast.Expression of p35S:OsPHT4s-GFP in rice protoplasts. The green signals indicate green fluorescent protein (GFP) and the red signals indicate autofluorescence of chlorophyll (the first six panels) and mcherry (Golgi marker) (the last two panels), respectively. In the last two panels, the GFP-OsPHT4;6-1/GFP-OsPHT4;6-2 construct and a Golgi marker were introduced into rice protoplasts for transient co-expression. BF, Bright field. Scale bars, 10 μm.

Fig. 4. OsPHT4 genes can confer Pi transport activity in yeast.Complementation of yeast mutant YP100 (Δpho84Δpho87Δpho89Δpho90Δpho91Δgit1) by the OsPHT4 genes. Equal volumes of 10-fold serial dilutions were applied to YNB(-P) (pH 5.5) medium with different Pi concentrations, and incubated at 30 ºC for 4 d. PHO84 is a high-affinity phosphate transporter and serves as a positive control, and empty vector as a negative control. YNB, Yeast nitrogen base without amino acids.

Supplemental Fig. 2. Analysis of OsPHT4 gene family promoter sequences.ABRE element, ABA responsive element, a cis-element involved in ABA response; P1BS element, PHR1 binding site, a cis-element enriched in phosphorus starvation induced gene promoters. The promoter length is 2500 bp for each gene.

Fig. 5. Dynamic changes of transcript levels of OsPHT4 genes during phosphate deprivation in shoots of rice seedlings. The expression level was represented as relative to the value under +P conditions. The wild-type plants were grown under Pi-replete conditions (200 mmol/L) for two weeks and then transferred to Pi starvation conditions (0 mmol/L). Shoot tissues were harvested from plants exposed to Pi-limiting conditions for 5 and 15 d, and the expression levels were compared with those detected under Pi-replete conditions. SQD2 was used to control the efficiency of phosphate deprivation treatments. Data are Mean ± SD (n = 3). *, P ≤ 0.05 and **, P ≤ 0.01 by the Student’s t-test.

Fig. 6. Dynamic changes of transcript levels of OsPHT4 genes in response to abscisic acid (ABA), salt and salicylic acid (SA) stresses in roots of rice seedlings. The expression level was represented as relative to the value at 0 h. Fourteen-day-old seedlings grown under normal conditions were exposed to different chemical treatments for various time periods. ABA, 100 mmol/L; NaCl, 100 mmol/L; SA, 500 μmol/L. OsbZIP23 was used to control the efficiency of ABA and NaCl treatments. OsWRKY45 was used to control the efficiency of SA treatments.Data are Mean ± SD (n = 3). *, P ≤ 0.05 and **, P ≤ 0.01 by the Student’s t-test.