# Functional PTB phosphate transporters are present in streptophyte algae and early diverging land plants

Clémence Bonnot, Helene H. Proust, Benoit Pinson, Florine P L Colbalchini, Alexis Lesly-Veillard, Holger Breuninger, Clément Champion, Alexander J Hetherington, Steven Kelly, Liam Dolan

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Clémence Bonnot, Helene H. Proust, Benoit Pinson, Florine P L Colbalchini, Alexis Lesly-Veillard, et al.. Functional PTB phosphate transporters are present in streptophyte algae and early diverging land plants. New Phytologist, 2017, 214 (3), pp.1158-1171. 10.1111/nph.14431. hal-02354932

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Functional PTB phosphate transporters are present in
streptophyte algae and early diverging land plants

Clémence Bonnot¹, Hélène Proust¹, Benoît Pinson²,³, Florine P. L. Colbalchini¹, Alexis Lesly-Veillard¹, Holger Breuninger¹, Clément Champion¹, Alexander J. Hetherington¹, Steven Kelly¹ and Liam Dolan¹

¹Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK; ²Centre National de la Recherche Scientifique (CNRS), UMR 5095 Institut de Biochimie et Génétique Cellulaire (IBGC), Bordeaux Cedex F-33077, France; ³Université de Bordeaux, Bordeaux F-33000, France

Author for correspondence:
Liam Dolan
Tel: +44 (0) 1865 275147
Email: Liam.Dolan@plants.ox.ac.uk

Received: 11 August 2016
Accepted: 15 December 2016

New Phytologist (2017) **214**: 1158–1171
**doi**: 10.1111/nph.14431

**Key words:** Coleochaete nitellarum, early diverging land plants, Marchantia polymorpha, Pi uptake, PTB Pi transporters, streptophyte algae.

Summary

* Two inorganic phosphate (Pi) uptake mechanisms operate in streptophytes and chlorophytes, the two lineages of green plants. PHOSPHATE TRANSPORTER B (PTB) proteins are hypothesized to be the Na⁺/Pi symporters catalysing Pi uptake in chlorophytes, whereas PHOSPHATE TRANSPORTER 1 (PHT1) proteins are the H⁺/Pi symporters that carry out Pi uptake in angiosperms. PHT1 proteins are present in all streptophyte lineages. However, Pi uptake in streptophyte algae and marine angiosperms requires Na⁺ influx, suggesting that Na⁺/Pi symporters also function in some streptophytes.

* We tested the hypothesis that Na⁺/Pi symporters exist in streptophytes.

* We identified PTB sequences in streptophyte genomes. Core PTB proteins are present at the plasma membrane of the liverwort *Marchantia polymorpha*. The expression of *M. polymorpha* core PTB proteins in the *Saccharomyces cerevisiae pho2* mutant defective in high-affinity Pi transport rescues growth in low-Pi environments. Moreover, levels of core PTB mRNAs of *M. polymorpha* and the streptophyte alga *Coleochaete nitellarum* are higher in low-Pi than in Pi-replete conditions, consistent with a role in Pi uptake from the environment.

* We conclude that land plants inherited two Pi uptake mechanisms - mediated by the PTB and PHT1 proteins, respectively - from their streptophyte algal ancestor. Both systems operate in parallel in extant early diverging land plants.

Introduction

Phosphorus is an essential macronutrient required for structural and metabolic functions in all living organisms. Phosphorus is absorbed largely as inorganic phosphate (Pi), through Pi transporters located at the plasma membrane of cells at the interface with the external environment. High-affinity and low-affinity Pi transporters catalyse Pi uptake from environments containing micromolar and millimolar concentrations of Pi, respectively (Werner et al., 1998; Harris et al., 2001; Werner & Kinne, 2001; Bucher, 2006; Dick et al., 2014). Chlorophytes and streptophytes are two monophyletic clades that constitute the green plant lineage. The land plants (also known as embryophytes) are a monophyletic group of streptophytes and the angiosperms are the most derived group of land plants (Ruhfel et al., 2014). Angiosperms absorb Pi through PHOSPHATE TRANSPORTER 1 (PHT1) plasma membrane H⁺/Pi symporters (Nussaume et al., 2011). The kinetics of Pi uptake in yeast mutants and plant protoplasts expressing plant PHT1 proteins have demonstrated that PHT1 genes encode either high-affinity or low-affinity H⁺/Pi symporters (Leggewie et al., 1997; Mitsukawa et al., 1997; Daram et al., 1998; Harrison et al., 2002; Rae et al., 2003; Liu et al.,

2008). Consistent with a role in Pi absorption from low-Pi environments, the steady state level of mRNA of most members of the PHT1 gene family is higher in plants grown in low-Pi than in Pi-replete environments, and in roots than in shoots (Mudge et al., 2002; Nussaume et al., 2011; Qin et al., 2012). The PHT1 proteins are the only plasma membrane Pi transporters identified in angiosperms (Ayadi et al., 2015). Furthermore, Pi uptake is 87% lower in the Atpht1;1 Atpht1;2 Atpht1;3 Atpht1;4 quadruple mutant than in wild-type (WT) *Arabidopsis thaliana*, suggesting that the PHT1 family is the major plasma membrane Pi transporter family in angiosperms (Ayadi et al., 2015).

Physiological evidence suggests that Na⁺/Pi symporters are involved in Pi uptake in algae and in some marine angiosperms. Na⁺ uptake is necessary for Pi influx in the streptophyte alga *Chara corallina* (Reid et al., 2000; Mimura et al., 2002) and stimulates Pi uptake in the marine angiosperm *Zostera marina* (Rubio et al., 2005) when grown in low-Pi conditions. This suggests that Na⁺/Pi symporters may also function as plasma membrane Pi transporters in some groups of streptophytes. Physiological analysis of membrane transport has demonstrated that Pi uptake involves an Na⁺/Pi symport mechanism in at least four chlorophytes (*Dunaliella salina*, *Ankistrodesmus braunii*, *Chlorella*
*pyrenoidosa, Hydrodictyon africanum* (Raven, 1984; Weiss *et al.*, 2001). It has been suggested that PHOSPHATE TRANSPORTER B (PTB) proteins, members of the Pi TRANSPORTER (PiT) protein family, are the Na⁺/Pi symporters involved in chlorophyte Pi uptake because of the sequence conservation and structural similarity of these proteins to the members of two other PiT subfamilies – the metazoan PiT and fungal PiT – known to be functional plasma membrane Na⁺/Pi symporters (Chung *et al.*, 2003; Kobayashi *et al.*, 2003; Li *et al.*, 2006, 2012; Moseley *et al.*, 2006; Guan *et al.*, 2008; Djouani-Tahri *et al.*, 2011). Consistent with the hypothesized Na⁺/Pi symporter activity is the observation that PTB proteins of the chlorophyte *Dunaliella viridis* increase the Na⁺ sensitivity of a yeast mutant with no plasma membrane Na⁺ export pumps (Li *et al.*, 2006). Moreover, the higher steady state level of most PTB mRNAs in low-Pi relative to Pi-replete conditions in the chlorophyte algae *D. viridis* (Li *et al.*, 2006; Guan *et al.*, 2008), *Ostreococcus taurii* (Djouani-Tahri *et al.*, 2011), *Tetraselmis chuii* (Chung *et al.*, 2003) and *Chlamydomonas reinhardtii* (Kobayashi *et al.*, 2003; Moseley *et al.*, 2006) supports the hypothesis that PTB proteins are involved in Pi uptake from the environment in chlorophytes. However, the Pi transport function of the PTB proteins has not been demonstrated and PTB sequences have not been identified in streptophytes.

As Pi uptake in chlorophyte and streptophyte algae and some aquatic angiosperms depends on the activity of Na⁺/Pi symporters, we set out to determine whether Na⁺/Pi symporters were encoded in the genomes of streptophytes whose life cycle is dependent on aquatic or semi-aquatic environments (streptophyte algae, early diverging land plants and aquatic angiosperms). Here, we report that genes encoding PTB proteins are present in the genomes of streptophytes (both algae and land plants), and that core PTB proteins pass through the secretory system to the plasma membrane in *Marchantia polymorpha*. We show that heterologous expression of *M. polymorpha* core PTB proteins is sufficient to restore Pi uptake in a *Saccharomyces cerevisiae* mutant with defective high-affinity Pi transport (Pinson *et al.*, 2009). Furthermore, the increased expression of streptophyte algae and early diverging land plant core *PTB* genes in low-Pi growth conditions supports the hypothesis that these proteins are involved in Pi uptake from the environment.

## Materials and Methods

### Sequence collection

The genome and transcriptome databases used to retrieve the nucleic and amino acid sequences are specified in Supporting Information Table S1. The GenBank accession numbers for the coding sequence (CDS) and corresponding proteins reported in this article are as follows: KX495367 (MpPTB1), KX495368 (MpPTB2), KX495369 (MpPTB3), KX495370 (MpPTB4), KX495371 (MpPTB5), KX495372 (MpPTB6), KX495373 (MpPTB7), KX495374 (MpPTB8), KX495375 (MpPHT2-1), KX495376 (CnPTB) and KX495377 (CnPHT2-1).

The amino acid sequences of ScPho89 (YBR296C) and NcPho4 (NCU09564) were used as queries in TBLASTN searches (Altschul *et al.*, 1990) for PiT sequences in the genomes and transcriptomes of 23 green plant species (species list in Table S1) and in the National Center for Biotechnology Information (NCBI) non-redundant nucleotide database. No *E*-value threshold was used; all hit sequences were systematically analysed as described hereafter. For each species, the retrieved transcripts were aligned using Multalin (Corpet, 1988) and compared with each other to eliminate duplicates and to assemble partial transcripts (annotated in Table S1). Amino acid sequences were predicted from transcripts using ExPASy translate (ExPASy, University of Geneva, Geneva, Switzerland) and from genomic sequences with Softberry FGENESH⁺ (Solovyev, 2007) using AtPHT2-1 and CrPTB2 as gene models. Reciprocal BLAST searches (Altschul *et al.*, 1990) were conducted on NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to verify whether the sequences belonged to the clade of interest. Nucleic acid and amino acid sequences were aligned (Figs S1, S2) using the iterative refinement strategy E-INSI (Katoh *et al.*, 2005) in MAFFT version 7 (Katoh & Standley, 2013). The alignments were manually adjusted with BIOEDIT v.7.1 (Ibis Biosciences, Carlsbad, CA, USA). Comparison of the nucleic acid and amino acid alignments allowed the identification of putative pseudogenes with interrupted open reading frames, and partial proteins resulting from partial transcripts or gene prediction errors (Table S1).

### Phylogenetic analyses and sequence nomenclature

To increase the resolution of the phylogenies, the pseudogenes and partial sequences were included in the analyses (Jiang *et al.*, 2014). The PiT amino acid alignments were trimmed manually with BIO-EDIT v.7.1 (Ibis Biosciences) to remove non-conserved regions (Figs S1, S2). The LG amino acid substitution model (Le & Gascuel, 2008) was estimated to be the best fit for the analysis of the PTB phylogenies using PROTTEST v.2.4 (Abascal *et al.*, 2005). ATGC PhyML was used to generate maximum-likelihood phylogenetic trees (Guindon *et al.*, 2010). Statistical support values of the embranchments were calculated with an approximate likelihood ratio test (aLRT) (Guindon *et al.*, 2010). Trees were visualized using FIGTREE (http://tree.bio.ed.ac.uk/software/figtree/). For each gene, the name used in the study and the name according to the Commission of Plant Nomenclature are given in Table S1.

### PiT amino acid sequence analysis

Percentages of similarity and identity between plant PiT proteins and ScPho89 were calculated using Sequence Identity And Similarity (SIAS) (http://imed.med.ucm.es/Tools/sias.html). Hydrophathy profiles and subcellular localizations were predicted using TMPRED (http://www.ch.embnet.org/software/TMPRED_form.html) and TARGETP (Emanuelsson *et al.*, 2007). Protein motifs were identified using MEME (Multiple Em for Motif Elicitation) (Bailey & Elkan, 1994) with a constraint of 30 conserved motifs. The presence of the motifs in streptophyte core PTB and PTB clade 4 proteins was assessed using MEME and the alignments.
Plant growth conditions

*Coleochaete nitellarum:* 1 g of WT strain culture (accession pro-
vided by the Skidmore Algal Collection of Professor David
Domozych), obtained from a single colony and grown in cell cul-
ture flasks (cell star, 250 ml, filter cap; Greiner Bio-One,
Kremsmünster, Austria) in 75 ml of liquid NaNO₃-BBM (Bold
Basal Medium (Nichols, 1973) supplemented with 1 g l⁻¹
NaNO₃, renewed every 6 wk), was transferred in cell culture
flasks (cell star, 50 ml, filter cap; Greiner Bio-One) in 25 ml of
liquid Pi-modified 3N-BBM + V (CCAP; Culture Collection of
Algae and Protozoa; Dunstaffnage Marine Laboratory, Oban,
UK) for 2 wk. 3N-BBM + V media with high- and low-Pi con-
tent were obtained by mixing the stock solutions according to the
manufacturer's instructions, except for K₂HPO₄ and KH₂PO₄,
which were added in identical amounts to the media to a final Pi
concentration of 0, 50, 500 or 1600 µM. K₂SO₄ was added to
each Pi-modified 3N-BBM + V medium to restore the final con-
centration of potassium to 2.15 mM. Algae were grown under a
cycle of 8 h: 16 h, dark: light (38 µmol m⁻² s⁻¹) at 23°C at
45 rpm.

*Marchantia polymorpha:* male (Takaragaike-1) and female (Takaragaike-2) (Ishizaki *et al.*, 2008) WT gemmae were propagated on sterile half-strength Johnson medium (Johnson *et al.*, 1957), pH 5.6, supplemented with 0.6% agar. One-week-old plants were transferred on low- or high-Pi modified half-strength Johnson medium (Saint-Marcoux *et al.*, 2015) for 2 wk. Plants were grown under continuous illumination (38 µmol m⁻² s⁻¹) at 23°C.

Coleochaete nitellarum de novo transcriptome assembly

The whole-plant *C. nitellarum* draft transcriptome was generated
by the sequencing of a strand-specific cDNA library with an
insert size of 500 bp using a paired-end read length of 2 × 100 bp
on Illumina Hiseq2000. Both the library preparation from 2 µg
RNA and the sequencing were performed by GATC Biotech Ltd
(Konstanz, Germany). For the assembly, raw reads were quality
trimmed using TRIMMOMATIC-0.32 (Bolger *et al.*, 2014),
BAYESHAMMER (SPADES-3.5.0) (Nikolenko *et al.*, 2013) and ALL-
PATHS-LG-4832 (Butler *et al.*, 2008). Ribosomal RNA was
removed using SORTMERNA-1.9 (Kopylova *et al.*, 2012). The qual-
ity trimmed reads were then normalized with a khmer size of 21
in KHMER-0.7.1 (Crusoe *et al.*, 2015). Normalized reads were
then assembled and scaffolded using SGA (Simpson & Durbin,
2012), SSPACE-v.3 (Boetzer *et al.*, 2011) and CAP3 (Huang &
Madan, 1999). Assembled scaffolds then underwent a final
correction step using PILON-1.6 (Walker *et al.*, 2014).

RNA extraction, cDNA synthesis, sequencing and
expression analysis

*Marchantia polymorpha* rhizoids and thalli were collected from
the same plants and flash frozen in liquid nitrogen. Total RNA
was extracted from ground frozen tissue with an RNeasy plant
mini kit (#74904; Qiagen, Venlo, the Netherlands) and DNase

treated on-column using DNase I (#79254; Qiagen). *Coleochaete nitellarum* samples were collected on 0.2-µm nylon sterile filters and flash frozen in liquid nitrogen. Total RNA was extracted from ground frozen tissue with a Zymo Research Direct-Zol RNA Miniprep kit (#R2051; ZymoResearch, Irvine, CA, USA) and DNase treated using Turbo DNase (#AM1907; Thermo Fisher Scientific, Waltham, MA, USA). cDNAs from *M. polymorpha* and *C. nitellarum* samples were synthesized from 1 µg of total RNA in a 20-µl reaction using 200 U of Protoscript II reverse transcriptase (#M0368S; NEB, Ipswich, MA, USA) and dT₁₇ oligos at 2 µM, and diluted in 180 µl of nuclease-free water. *Marchantia polymorpha* and *C. nitellarum* sequences were verified by PCR amplification (primers listed in Table S2) and Sanger sequencing (Source Bioscience, Nottingham, UK).

Quantitative PCR experiments were performed as described pre-
viously (Saint-Marcoux *et al.*, 2015) for *M. polymorpha* and using
20 µl of reaction volume containing 1 µM of forward and reverse
primers, 10 µl of 2 × SensiFAST Sybr-Green mHi-ROX master
mix (#BIO-92020; Bioline, London, UK) and 5 µl of diluted
cDNA for *C. nitellarum*. Amplification experiments were carried
out using a 7300 Applied Biosystems thermocycler (Thermo Fisher
Scientific). Data analysis was performed as described previously
(Saint-Marcoux *et al.*, 2015). Reference genes were Mp*ACT* and
Mp*SAND* for *M. polymorpha* (Saint-Marcoux *et al.*, 2015), and
Cn*EFIα* for *C. nitellarum* (primers listed in Table S2).

Vector construction

The CDSs encoding the yellow fluorescent protein (YFP) and
MpPTB6 proteins were amplified from a published construct
(Breuninger et al., 2016) and from cDNA, respectively, for con-
struction of the pOsAct1:MpPTB6-YFP:Term and pOsAct1:YFP:
Term binary vectors using the high-fidelity polymerase Phusion
(#F-530L; Thermo Fisher Scientific) with the primers listed in
Table S2. The reverse primer for MpPTB6 CDS amplification
and the forward primer for YFP CDS amplification for the trans-
lational fusion are partially complementary and contain a
sequence encoding a peptide linker between MpPTB6 and YFP.
The assembly of the amplicons of the MpPTB6 and YFP CDSs
in one MpPTB6-YFP CDS was carried out by PCR using the
high-fidelity polymerase Phusion (#F-530L; Thermo Fisher Sci-
entific) with the forward primer for MpPTB6 amplification and
the reverse primer for YFP amplification. MpPTB6-YFP and
YFP CDSs were cloned into pCR8-GW-TOPO (#K2500-20;
Thermo Fisher Scientific) to establish Gateway™ entry vectors.
These were subsequently recombined, using the LR reaction
(#12538-200; Thermo Fisher Scientific), with the destination
vector pCambia pOsAct1:GW:Term (Proust et al., 2016) con-
taining the rice ACTIN1 promoter, pOsAct1, driving the consti-
tutive expression in M. polymorpha (Breuninger et al., 2016).

MpPTB4, MpPTB6 and MpPTB7 CDSs were amplified from
cDNA for the construction of the yeast expression/shuttle vectors
using the high-fidelity polymerase Phusion (#F-530L; Thermo
Fisher Scientific) with the adaptor primers listed in Table S2. CDSs
were cloned into the pGEM®-T easy vector (Promega, Madison,
WI, USA) and subcloned into the pCM189 vector behind a
tetracycline repressible promoter (Gari *et al.*, 1997). Subclonings of MpPTB6 and MpPTB7 in *pCM189* were performed between BamHⅠ and PstⅠ restriction sites, using the BamHⅠ and PstⅠ restriction sites designed at the 5' end of the adaptor primers. Subcloning of MpPTB4 in *pCM189* was performed between NotⅠ and PstⅠ restriction sites using the NotⅠ and PstⅠ restriction sites of the Multicloning-site of the PGemT-easy vector.

*Saccharomyces cerevisiae* strains, transformation and culture

The *S. cerevisiae* strain Δ*pho2* Y2092 (*Matα his3Δ1 leu2Δ0 ura3Δ0 pho2::KanMX6*; B. Pinson, unpublished) (*ScΔpho2*) was transformed (Chen *et al.*, 1992) with *pCM189* vectors expressing the MpPTB4, MpPTB6 or MpPTB7 CDS or the *S. cerevisiae* *ScPho84* or *ScPho89* CDS (Pinson *et al.*, 2004). The Δ*pho2* Y2092 and *ScPH02* WT Y178 (*Matα his3Δ1 leu2Δ0 ura3Δ0* derived from BY4742; Euroscarf, Frankfurt, Germany) strains were transformed with the cognate *pCM189* empty vector. A drop test was used to compare the growth of the different transformed strains, as described previously (Pinson *et al.*, 2004). Drops of cell dilutions were spotted onto freshly prepared plates of a modified SD medium containing 0.57% of yeast nitrogen base with (NH₄)₂SO₄, but without Pi and NaCl (#114027812; MP Biomedical, Santa Ana, CA, USA), 2% glucose, 2 mM NaCl and 2.5% agar, buffered at pH 6 with 50 mM phthalic acid and supplemented with tryptophan (0.2 mM) and adenine (0.3 mM). The Pi concentration was adjusted using KHPO₃. When required, doxycycline (10 mg l⁻¹) was used to repress the expression of the various CDSs under the tetracycline-repressible promoter (Gari *et al.*, 1997). Cultures were grown at 30°C for 11 d before scanning.

*Marchantia polymorpha* transformation and confocal microscopy imaging

*Marchantia polymorpha* sporelings were transformed with one of four binary vectors: *pMpSYP12A:mCitrine-MpSYP12A:Term* (Kanazawa *et al.*, 2016), *p35S:YFP:HDEL:Term* (Teh & Moore, 2007), *pOsAct1:YFP:Term* and *pOsAct1:MpPTB6-YFP:Term* using a published protocol (Proust *et al.*, 2016). Images were captured using an upright confocal microscope (Leica DM6000CS; Leica, Wetzlar, Germany) and Leica Advance Fluorescence Leica application suite software (LAS AF). Excitation of the fluorophores and chlorophyll was performed with an argon laser (514 nm) at 29% power. Emissions were acquired at 525–571 nm for YFP and mCitrine, and at 696–761 nm for chlorophyll. Pinhole *m* = 111.5 µm and pinhole airy = 1. Cell and tissue images were obtained with ×20 and ×50 lenses.

Results

Identification of PiT-encoding genes in chlorophytes and streptophytes

Plant PiT proteins, which comprise members of both the PTB and PHOSPHATE TRANSPORTER 2 (PHT2) subfamilies, are

homologues of the fungal PiT Pi transporters ScPho89 of *S. cerevisiae* and NcPho4 of *Neurospora crassa*. The PTB and PHT2 protein subfamilies are distinct phylogenetically (Versaw & Harrison, 2002; Chung *et al.*, 2003; Kobayashi *et al.*, 2003; Zhao *et al.*, 2003; Moseley *et al.*, 2006; Tyra *et al.*, 2007; Djouani-Tahri *et al.*, 2011; Li *et al.*, 2012). Moreover, members of the PHT2 protein subfamily are H⁺/Pi symporters located at the chloroplast envelope with no role in Pi uptake to the cell (Versaw & Harrison, 2002; Zhao *et al.*, 2003). There are no published amino acid or nucleic motifs specific to the PHT2 or PTB subfamilies. Therefore, to identify new chlorophyte and streptophyte *PTB* sequences, we first identified the *PiT* (*PHT2* and *PTB*) sequences in the genomes and transcriptomes of 23 taxa representative of the green plant lineage by searching for protein sequences similar to ScPho89 and NcPho4 using the TBLASTN algorithm (Altschul *et al.*, 1990). We identified 74 sequences (Fig. 1; Table S1). Their predicted amino acid sequences were 42–87% similar and 15–36% identical to ScPho89 (Table S1) and contained the PiT signature motifs: the PiT N- and C-terminal GANDVAN domains (G-A/S/F-N/H-D-V/T-A-N-A/S and G-A/G-N/G-D-V-x-x-x) and the two glutamate (E⁵⁵ and E⁵⁷⁵ in HsPit2) and serine (S¹¹³ and S⁵⁹³ in HsPit2) PiT conserved residues (Salaün *et al.*, 2001, 2004; Bøttger & Pedersen, 2002, 2005; Bøttger *et al.*, 2006) (Fig. S1). These data suggest that all 74 sequences identified are either PiT-encoding genes or PiT-related pseudo-genes in the case of 14 partial sequences. They include 60 new *PiT* sequences, the 10 published *C. reinhardtii* PTB proteins (Kobayashi *et al.*, 2003; Moseley *et al.*, 2006) and four published PHT2 proteins from the angiosperm *A. thaliana* (Versaw & Harrison, 2002), *Solanum lycopersicum* (Chen *et al.*, 2007), *Medicago truncatula* (Zhao *et al.*, 2003) and *Oryza sativa* (Liu *et al.*, 2011) (Figs 1, S1). Taken together, these data indicate that PiT proteins are present in both chlorophytes and streptophytes, including streptophyte algae and land plants.

PTB proteins are present in both chlorophytes and streptophytes

We used maximum-likelihood statistics (Guindon *et al.*, 2010) to determine the phylogenetic relationships between the 60 new *PiT* sequences identified in this survey and the PHT2 and PTB PiT subfamilies. The topology of the phylogenetic tree showed that the plant PiT sequences form two monophyletic groups corresponding to the PHT2 and PTB subfamilies (Figs 2, S3). The PHT2 subfamily is sister to the bacterial PiT proteins and, together with the animal PiT proteins, constitutes a sister group to the PTB subfamily (Figs 2, S3) (Tyra *et al.*, 2007). The PHT2 subfamily (supported by an aLRT value of 1) includes 22 of the new PiT sequences and the four published angiosperm PHT2 proteins (Versaw & Harrison, 2002; Zhao *et al.*, 2003; Chen *et al.*, 2007; Liu *et al.*, 2011) (Figs 2, S3). *PHT2* sequences were identified in all the streptophyte taxa used in this study, but not in chlorophytes, suggesting that PHT2 proteins are streptophyte specific.

The remaining 38 new PiT sequences are members of the monophyletic PTB subfamily (supported by an aLRT value of
0.75) which form four different monophyletic clades (Figs 2, S3). The largest monophyletic clade (supported by an aLRT value of 0.99) comprises 34 sequences that include the 12 published chlorophyte PTB proteins present in our data (the *C. reinhardtii* CrPTB1–CrPTB9 and CrPTB12, and the *D. viridis* DvSPT1 and DvSPT2) (Kobayashi *et al.*, 2003; Li *et al.*, 2006; Moseley *et al.*, 2006; Guan *et al.*, 2008). This clade was therefore designated the core PTB clade. Core PTB proteins were identified in both chlorophytes and streptophytes, including the streptophyte

algae *Klebsormidium flaccidum* and *Coleochaete nitellarum*, and the early diverging land plants *M. polymorpha* (a liverwort), *Physcomitrella patens* and *Encalypta streptocarpa* (two mosses), and *Nothoceros vincentianus* and *Phaeoceros carolinianus* (two hornworts). There are no tracheophyte members of the core PTB clade. The second monophyletic PTB clade (PTB clade 2) forms an algal-specific clade (supported by an aLRT value of 1) and comprises sequences from the chlorophyte *Coccomyxa subellipsoidea* and the streptophyte *K. flaccidum*. The third

<table><caption>**Fig. 1** Number and type of PiT sequences identified in 23 representative species of green plant. The total number of PHOSPHATE (Pi) TRANSPORTER (PiT) sequences identified (PiT identified) in the genomic and transcriptomic databases of 23 green plant taxa can be divided into three categories: (1) the PHOSPHATE TRANSPORTER B (PTB) sequences that have been published previously (Published PTB); (2) the PHOSPHATE TRANSPORTER 2 (PHT2) sequences that have been published previously (Published PHT2); (3) the new sequences identified (New PiT). The number of putative pseudo-genes or partial sequences among the sequences is given in parentheses. The taxa are arranged according to the green plant phylogeny. The dashed branches of the tree indicate the paraphyletic clades (charophyte and bryophyte).</caption><thead><tr><th></th><th>O. lucimarinus</th><th>C. subellipsoidea</th><th>C. reinhardtii</th><th>K. flaccidum</th><th>C. nitellarum</th><th>M. polymorpha</th><th>P. patens</th><th>E. streptocarpa</th><th>P. carolinianus</th><th>S. moelendoffii</th><th>H. lucidula</th><th>D. conjugata</th><th>C. acrostichoides</th><th>P. abies</th><th>P. taeda</th><th>S. polyrhiza</th><th>Z. marina</th><th>Z. moys</th><th>O. sativa</th><th>M. truncatula</th><th>A. thalina</th><th>Total</th></tr><tr><th>PiT identified</th><th>2</th><th>3</th><th>11(1)</th><th>8(2)</th><th>2</th><th>9</th><th>4(1)</th><th>4(1)</th><th>3(2)</th><th>8(4)</th><th>2</th><th>2(1)</th><th>3</th><th>3(1)</th><th>2</th><th>1(1)</th><th>1</th><th>1</th><th>1</th><th>1</th><th>1</th><th>1</th><th>74(14)</th></tr></thead><tbody><tr><th>Published PTB</th><td>-</td><td>-</td><td>10</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>10</td></tr><tr><th>Published PHT2</th><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>1</td><td>1</td><td>1</td><td>4</td></tr><tr><th>New PiT (PTB and PHT2)</th><td>2</td><td>3</td><td>1(1)</td><td>8(2)</td><td>2</td><td>9</td><td>4(1)</td><td>4(1)</td><td>3(2)</td><td>8(4)</td><td>2</td><td>2(1)</td><td>3</td><td>3(1)</td><td>2</td><td>1(1)</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>60 (14)</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>**Fig. 2** Schematic representation of the maximum-likelihood phylogeny of the green plant PiT sequences. The phylogeny was produced using the multiple amino acid alignment of the plant PHOSPHATE (Pi) TRANSPORTER (PiT) sequences. Three metazoan PiT (DmPit, HsPit1 and HsPit2), three bacterial PiT (TePit, EcPitA and EcPitB) and two fungal PiT (ScPho89 and NcPho4) proteins were used as outgroups. Sequence names consist of the species code (first letter of the genus and first letter of the species name, or first letter of the genus and two first letters of the species name for *Zostera marina*) and gene name (as published or given according to the published sequences with which they grouped in this phylogenetic analysis). The major nodes of the tree are marked by a point (red for the PiT subfamilies, blue for the PHOSPHATE TRANSPORTER B (PTB) clades and black for others). The approximate likelihood ratio test (aLRT) support values are given for the major nodes. *The published PHOSPHATE TRANSPORTER 2 (PHT2) or PTB sequences.</figcaption></figure>
monophyletic PTB clade (PTB clade 3; supported by an aLRT value of 1) comprises a pair of paralogous sequences from the chlorophyte *Ostreococcus lucimarinus*. Its separation from PTB clade 2 is poorly resolved (supported by an aLRT value of 0.37). The fourth monophyletic clade (PTB clade 4, supported by an aLRT value of 1) is land plant specific and sister to all other PTB clades. It comprises sequences from the hornworts *N. vincentianus* and *P. carolinianus*, the lycophytes *Selaginella moelendorfii* and *Huperzia lucidula*, the monilophytes *Dipteris conjugata* and *Cryptogamma acrostichoides*, and the gymnosperm *Picea abies*. No PTB clade 4 sequences were identified in liverworts, mosses or angiosperms (Figs 2, S3).

An analysis of the PiT sequences that included all plant PiT proteins (89 sequences) identified in the NCBI non-redundant nucleotide database confirmed all four monophyletic clades and the topology of the PTB subfamily (Fig. S4). This analysis did not identify new core PTB members among these sequences, which confirmed that core PTB proteins do not exist in tracheophytes. Seventeen new PTB clade 4 members were identified in several angiosperm lineages, including basal angiosperms, monocots and eudicots, demonstrating that secondary losses can explain the absence of PTB clade 4 members in some gymnosperm (*P. taeda*) and angiosperm species. This also suggests that secondary loss is responsible for the absence of PTB clade 4 proteins in liverworts, mosses and streptophyte algae. Together, these data demonstrate that PTB proteins are present in both chlorophytes and streptophytes and that core PTB proteins are present in streptophyte algae and early diverging land plants (liverworts, mosses and hornworts).

## The structure of core PTB proteins is conserved between streptophytes and chlorophytes

To evaluate the degree of structural and functional conservation between chlorophyte and streptophyte core PTB proteins, we compared their predicted topology, subcellular location and conserved motifs (Tables 1–3). The published core PTB proteins of chlorophytes have 12 transmembrane domains (TMDs) and a large hydrophilic loop between the seventh and eighth TMDs (Chung *et al.*, 2003; Kobayashi *et al.*, 2003; Li *et al.*, 2006, 2012). TMPRED hydropathy profile prediction software (Hofmann & Stoffel, 1993) predicted a large hydrophilic loop between the seventh and eighth TMDs in all streptophyte core PTB proteins, as in the chlorophyte core PTB proteins (Table 1). A large glycine- and glutamine-rich region inserted in the hydrophilic loop of two core PTB proteins (CrPTB1 and CrPTB8) of the chlorophyte *C. reinhardtii* (Fig. S1) is responsible for the difference in their predicted hydropathy profile from all other core PTB proteins (Kobayashi *et al.*, 2003; Moseley *et al.*, 2006). These data indicate that the topology of core PTB proteins is conserved between streptophytes and chlorophytes.

An N-terminal signal peptide targets proteins to the secretory pathway; proteins are inserted into the endoplasmic reticulum (ER) and either remain in the ER or move to the plasma membrane, vacuole or endomembrane compartments (Chrispeels & Raikhel, 1992). There is a signal peptide in core PTB proteins of

the chlorophyte *C. reinhardtii* (Moseley *et al.*, 2006). Using TargetP software (Emanuelsson *et al.*, 2007), which predicts subcellular location by detecting subcellular targeting motifs, we discovered a signal peptide at the N-terminus of all streptophyte core PTB proteins (Table 2). Therefore, streptophyte core PTB proteins are probably targeted to the secretory pathway. In contrast, TargetP did not identify a signal peptide in any members of the PTB clade 4, except for the *S. moelendorfii* PTB (SmPTB) (Table S3). This suggests that, although core PTB proteins traffic to the secretory pathway, the PTB clade 4 proteins do not.

To test the hypothesis that core PTB proteins traffic to the plasma membrane through the secretory pathway, we expressed an MPPTB6-YFP fusion protein in *M. polymorpha*. YFP fluorescence location was identical to that of the plasma membrane marker mCitrine-MpSYP12A (Kanazawa *et al.*, 2016) and the ER marker YFP-HDEL (Teh & Moore, 2007) in 25% and 75%, respectively, of plants expressing MPPTB6-YFP (Fig. S5). This is consistent with the hypothesis that MPPTB6-YFP moves through the secretory pathway to reach the plasma membrane.

<table><caption>**Table 1** TMPRED predictions of the hydrophilic loop position of core PHOSPHATE TRANSPORTER B (PTB) proteins</caption><thead><tr><th></th><th>Species</th><th>Protein</th><th>TM number</th><th>Hydrophilic loop after</th></tr></thead><tbody><tr><td rowspan="15">Chlorophytes</td><td rowspan="15">Chlamydomonas reinhardtii</td><td>CrPTB1*</td><td>14</td><td>TMD10</td></tr><tr><td>CrPTB2</td><td>12</td><td>TMD7</td></tr><tr><td>CrPTB3</td><td>12</td><td>TMD7</td></tr><tr><td>CrPTB4</td><td>11</td><td>TMD7</td></tr><tr><td>CrPTB5</td><td>11</td><td>TMD7</td></tr><tr><td>CrPTB6</td><td>12</td><td>TMD7</td></tr><tr><td>CrPTB7</td><td>12</td><td>TMD7</td></tr><tr><td>CrPTB8 *</td><td>12</td><td>TMD8</td></tr><tr><td>CrPTB9</td><td>11</td><td>TMD7</td></tr><tr><td>CrPTB12</td><td>12</td><td>TMD7</td></tr><tr><td>Dunaliella viridis</td><td>DvSPT1</td><td>11</td><td>TMD7</td></tr><tr><td>DvSPT2</td><td>11</td><td>TMD7</td></tr><tr><td>Coccomyxa subellipsoidea</td><td>CsPTB1</td><td>12</td><td>TMD7</td></tr><tr><td rowspan="12">Streptophytes</td><td>Klebsormidium flaccidum</td><td>KfPTB1</td><td>13</td><td>TMD7</td></tr><tr><td>KfPTB2</td><td>13</td><td>TMD7</td></tr><tr><td>Coleochaete nitellarum</td><td>CnPTB</td><td>12</td><td>TMD7</td></tr><tr><td rowspan="8">Marchantia polymorpha</td><td>MpPTB1</td><td>12</td><td>TMD7</td></tr><tr><td>MpPTB2</td><td>11</td><td>TMD7</td></tr><tr><td>MpPTB3</td><td>12</td><td>TMD7</td></tr><tr><td>MpPTB4</td><td>12</td><td>TMD7</td></tr><tr><td>MpPTB5</td><td>12</td><td>TMD7</td></tr><tr><td>MpPTB6</td><td>12</td><td>TMD7</td></tr><tr><td>MpPTB7</td><td>12</td><td>TMD7</td></tr><tr><td>MpPTB8</td><td>11</td><td>TMD7</td></tr><tr><td rowspan="3">Physcomitrella patens</td><td>PpPTB1</td><td>12</td><td>TMD7</td></tr><tr><td>PpPTB2</td><td>11</td><td>TMD7</td></tr><tr><td>EsPTB1</td><td>12</td><td>TMD7</td></tr><tr><td rowspan="2">Encalypta streptocarpa</td><td>EsPTB2</td><td>11</td><td>TMD7</td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table>

The total number of predicted transmembrane domain (TM number) and the transmembrane domain after which TMPRED predicts the long hydrophilic loop (Hydrophilic loop after) is given for each core PTB protein with a complete sequence.

*Proteins with a glycine- and glutamine-rich region inserted in the hydrophilic loop.
There are no ER retention signals (KDEL or HDEL) (Chrispeels & Raikhel, 1992) at the N- or C-termini of any core PTB proteins (Figs S1, S6), suggesting that the retention of MpPTB6-YFP at the ER is an artefact caused by the over-expression or misfolding of the fusion protein in transgenic plants. Nevertheless, the absence of MpPTB6-YFP at the tonoplast supports the hypothesis that core PTB proteins are not located in this membrane.

We used MEME (Bailey & Elkan, 1994), a protein family motif finder software, to identify protein motifs that distinguish the core PTB proteins from other members of the PiT family. By analysing the sequence of the four published angiosperm PHT2 proteins (Versaw & Harrison, 2002; Zhao *et al.*, 2003; Chen *et al.*, 2007; Liu *et al.*, 2011), 12 published chlorophyte core PTB proteins (Kobayashi *et al.*, 2003; Li *et al.*, 2006; Moseley *et al.*, 2006; Guan *et al.*, 2008), fungal PiT proteins ScPho89 and NcPho4 (Mann *et al.*, 1989; Martinez & Persson, 1998), metazoan PiT proteins DmPit (from *Drosophila melanogaster*) (Bergwitz *et al.*, 2012), HsPit1 and HsPit2 (from *Homo sapiens*) (O'Hara *et al.*, 1990; Van Zeijl *et al.*, 1994), and bacterial PiT proteins TePit (NK55_00390 from *Thermosynechococcus elongatus*) (Bøttger & Pedersen, 2005), EcPitA and EcPitB (from *Escherichia coli*) (Harris *et al.*, 2001), MEME identified 18 conserved motifs distributed among the PiT subfamilies (Table 3; Fig. S6). Five motifs (motifs 1–5) were present in all PiT proteins. They contain the PiT signature GANDVAN domains

(motifs 1 and 4) and the PiT signature residues (motifs 2 and 5). One motif (motif 6) was common to the fungal PiT and core PTB proteins, but absent from the metazoan PiT, bacterial PiT and PHT2 proteins. Seven motifs (motifs 7–13) were specific to the core PTB proteins and five motifs (motifs 14–18) were specific to the PHT2 proteins. The analysis of the streptophyte core PTB amino acid sequences showed that all motifs identified in the sequence of the chlorophyte core PTB proteins (motifs 1–13) were present in the streptophyte core PTB proteins (Table 3; Fig. S6). None of the motifs specific to the PHT2 subfamily was found in streptophyte core PTB proteins. The conservation of the protein motifs between core PTB proteins of chlorophytes and streptophytes suggests that the core PTB proteins are structurally similar in the two lineages of green plants. By contrast, of the five motifs present in the PTB clade 4 proteins (motifs 1, 3, 4, 6 and 10), four were absent in the seed plant PTB clade 4 sequences (Table S4; Fig. S6), including the GANDVAN domains (motifs 1 and 4) and the glutamate PiT signature residues (E⁵⁵ and E⁵⁷⁵ in HsPit2), both necessary for the Pi transport function of fungal and metazoan PiT proteins (Bøttger & Pedersen, 2002, 2005; Bøttger *et al.*, 2006; Andersson *et al.*, 2012). The loss of these motifs suggests that the function of PTB clade 4 proteins changed during the course of land plant evolution.

Taken together, these results imply that there has been little sequence divergence of the core PTB proteins since chlorophytes

<table><caption>**Table 2** TargetP prediction of subcellular targeting of core PHOSPHATE TRANSPORTER B (PTB) proteins</caption><thead><tr><th rowspan="2"></th><th rowspan="2">Species</th><th rowspan="2">Protein</th><th colspan="4">TARGETP score</th><th rowspan="2">Prediction</th></tr><tr><th>Chloroplast</th><th>Mitochondrion</th><th>Secretory pathway</th><th>Other</th></tr></thead><tbody><tr><td rowspan="12">Chlorophytes</td><td rowspan="12">Chlamydomonas reinhardtii</td><td>CrPTB1</td><td>0.008</td><td>0.019</td><td>0.953</td><td>0.06</td><td>Secreted (RC1)</td></tr><tr><td>CrPTB2</td><td>0.004</td><td>0.008</td><td>0.913</td><td>0.395</td><td>Secreted (RC3)</td></tr><tr><td>CrPTB3</td><td>0.004</td><td>0.007</td><td>0.894</td><td>0.475</td><td>Secreted (RC3)</td></tr><tr><td>CrPTB4</td><td>0.002</td><td>0.012</td><td>0.979</td><td>0.309</td><td>Secreted (RC2)</td></tr><tr><td>CrPTB5</td><td>0.002</td><td>0.013</td><td>0.982</td><td>0.28</td><td>Secreted (RC2)</td></tr><tr><td>CrPTB6</td><td>0.011</td><td>0.015</td><td>0.943</td><td>0.339</td><td>Secreted (RC2)</td></tr><tr><td>CrPTB7</td><td>0.011</td><td>0.006</td><td>0.899</td><td>0.304</td><td>Secreted (RC3)</td></tr><tr><td>CrPTB8</td><td>0.011</td><td>0.029</td><td>0.688</td><td>0.108</td><td>Secreted (RC3)</td></tr><tr><td>CrPTB9</td><td>0.008</td><td>0.013</td><td>0.958</td><td>0.264</td><td>Secreted (RC2)</td></tr><tr><td>CrPTB12</td><td>0.008</td><td>0.014</td><td>0.957</td><td>0.243</td><td>Secreted (RC2)</td></tr><tr><td>KfPTB1</td><td>0.004</td><td>0.026</td><td>0.909</td><td>0.117</td><td>Secreted (RC2)</td></tr><tr><td>KfPTB2</td><td>0.006</td><td>0.224</td><td>0.687</td><td>0.098</td><td>Secreted (RC3)</td></tr><tr><td>Coleochaete nitellarum</td><td>CnPTB</td><td>0.016</td><td>0.019</td><td>0.889</td><td>0.114</td><td>Secreted (RC2)</td></tr><tr><td rowspan="10">Streptophytes</td><td rowspan="10">Klebsormidium flaccidum</td><td>MpPTB1</td><td>0.015</td><td>0.011</td><td>0.916</td><td>0.085</td><td>Secreted (RC1)</td></tr><tr><td>MpPTB2</td><td>0.034</td><td>0.008</td><td>0.801</td><td>0.259</td><td>Secreted (RC3)</td></tr><tr><td>MpPTB3</td><td>0.022</td><td>0.008</td><td>0.872</td><td>0.187</td><td>Secreted (RC2)</td></tr><tr><td>MpPTB4</td><td>0.02</td><td>0.009</td><td>0.875</td><td>0.179</td><td>Secreted (RC2)</td></tr><tr><td>MpPTB5</td><td>0.017</td><td>0.008</td><td>0.895</td><td>0.102</td><td>Secreted (RC2)</td></tr><tr><td>MpPTB6</td><td>0.031</td><td>0.009</td><td>0.796</td><td>0.265</td><td>Secreted (RC3)</td></tr><tr><td>MpPTB7</td><td>0.004</td><td>0.011</td><td>0.956</td><td>0.156</td><td>Secreted (RC1)</td></tr><tr><td>MpPTB8</td><td>0.012</td><td>0.01</td><td>0.902</td><td>0.143</td><td>Secreted (RC2)</td></tr><tr><td>PpPTB1</td><td>0.008</td><td>0.011</td><td>0.932</td><td>0.23</td><td>Secreted (RC2)</td></tr><tr><td>PpPTB2</td><td>0.006</td><td>0.011</td><td>0.934</td><td>0.154</td><td>Secreted (RC2)</td></tr><tr><td rowspan="2">Encalypta streptocarpa</td><td>EsPTB1</td><td>0.011</td><td>0.010</td><td>0.829</td><td>0.285</td><td>Secreted (RC3)</td></tr><tr><td>EsPTB2</td><td>0.026</td><td>0.009</td><td>0.743</td><td>0.310</td><td>Secreted (RC4)</td></tr><tr><td>Phaeoceros carolinianus</td><td>PcPTB4*</td><td>0.022</td><td>0.007</td><td>0.868</td><td>0.195</td><td>Secreted (RC2)</td></tr></tbody></table>

TARGETP prediction is given with a reliability class (RC) indicated, from 1 to 5, where '1' indicates the strongest prediction for streptophyte core PTB proteins with a complete sequence or with a partial sequence* if the N-terminus of the protein is present.
and streptophytes last shared a common ancestor. By contrast, PTB clade 4 proteins diverged considerably during the course of land plant evolution and the loss of the GANDVAN domains and conserved PiT residues suggests that they may not be Pi transporters.

### Expression of *M. polymorpha* core PTB proteins in the *S. cerevisiae* pho2 mutant rescues the *ScΔpho2* growth defect

The *S. cerevisiae* pho2 (*ScΔpho2*) mutant encodes a defective *ScPhO2* transcription factor required for the expression of the two *S. cerevisiae* high-affinity Pi transporters: *ScPho84* (homologue of plant PHT1 Pi transporters) and *ScPho89* (homologue of plant PTB Pi transporters) (Bun-Ya *et al.*, 1991; Martinez & Persson, 1998; Pinson *et al.*, 2009). The inability of *ScΔpho2* mutants to express high-affinity Pi transporters (*ScPho84* and *ScPho89*) means that growth is impaired in low-Pi environments (< 500 µM), but normal at Pi concentrations higher than 500 µM because of the constitutive expression of yeast low-affinity Pi transporters (Fig. 3). To determine whether the *M. polymorpha* core PTB proteins have a Pi transporter function, we tested the ability of MpPTB4, MpPTB6 and MpPTB7 proteins to rescue the *ScΔpho2* growth defect on low-Pi media.

Transformation of *ScΔpho2* mutants with constructs expressing MpPTB4, MpPTB6 or MpPTB7 rescued the *ScΔpho2* growth defect to varying degrees (Fig. 3). No growth was observed in any strain in the absence of Pi. At Pi concentrations equal to or greater than 250 µM, the expression of MpPTB4 partially rescued the *ScΔpho2* growth defect. At 500 µM of Pi, the expression of MpPTB6 and MpPTB7 partially rescued the

*ScΔpho2* growth defect. This suggests that the expression of MpPTB4, MpPTB6 and MpPTB7 proteins in the *ScΔpho2* mutant restores Pi transport. As controls, expression of the yeast high-affinity Pi transporters *ScPho84* or *ScPho89* in *ScΔpho2* completely rescued the mutant growth defect at a Pi concentration equal to or greater than 250 µM. The addition of doxycycline – a repressor of the tetracycline-responsive promoter used to express the transporter genes (Gari *et al.*, 1997) – blocked the growth rescue by MpPTB4, MpPTB6, MpPTB7, *ScPho84* and *ScPho89*. This indicates that the restoration of *ScΔpho2* growth is a result of the expression of the Pi transporters introduced on the expression plasmid. Taken together, these data are consistent with the hypothesis that MpPTB4, MpPTB6 and MpPTB7 are functional Pi transporters.

### The expression of the core *PTB* genes is induced in low-Pi environments in the streptophyte alga *C. nitellarum* and the liverwort *M. polymorpha*

In angiosperms, the steady state level of most PHT1 mRNAs is higher in low-Pi than in high-Pi environments, and in roots than in shoots, consistent with their role in Pi uptake from low-Pi environments (Mudge *et al.*, 2002; Nussaume *et al.*, 2011; Qin *et al.*, 2012). Similarly, the steady state level of most chlorophyte PTB mRNAs is also higher in low-Pi than in high-Pi environments (Chung *et al.*, 2003; Kobayashi *et al.*, 2003; Li *et al.*, 2006; Moseley *et al.*, 2006; Djouani-Tahri *et al.*, 2011). To determine whether the expression of genes encoding the core PTB proteins is regulated by Pi availability in streptophyte algae and early diverging land plants, we measured the steady state levels of core PTB mRNAs in the streptophyte alga *C. nitellarum* and the

<table><caption>**Table 3** Conserved motifs of PHOSPHATE (Pi) TRANSPORTER (PiT) subfamilies identified by MEME (Multiple Em for Motif Elicitation)</caption><thead><tr><th>Motif</th><th>AA</th><th>Sequence</th><th>PHT2</th><th>Bacterial PiT</th><th>Metazoan PiT</th><th>Fungal PiT</th><th>Chlorophyte core PTB</th><th>Streptophyte core PTB</th></tr></thead><tbody><tr><td>Motif 1</td><td>14</td><td>GaNDVANafgTSVg</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 2</td><td>15</td><td>IpvStTHxixGaxxG</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 3</td><td>6</td><td>SWfxSP</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 4</td><td>14</td><td>FaHGaNDVxnaGP</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 5</td><td>34</td><td>TPxrGfxaElaxaASxxGLPxStTG</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 6</td><td>12</td><td>HaxAEVFdPxtE</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 7</td><td>8</td><td>FpyxxGxV</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 8</td><td>12</td><td>kaAWVxaxxaG</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 9*</td><td>8</td><td>KxaaxxGx</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 10</td><td>12</td><td>AAxFAxGayaPS</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 11</td><td>7</td><td>kdxxkYe</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 12</td><td>5</td><td>INxxI</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 13</td><td>8</td><td>nxSxxtxG</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Yes</td><td>Yes</td></tr><tr><td>Motif 14</td><td>27</td><td>MAxAFHISsRtAxalSixIAfAALtxP</td><td>Yes</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Motif 15</td><td>19</td><td>KxKxLsYxTLLfGFYMAWNI</td><td>Yes</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Motif 16</td><td>29</td><td>KCIRRFVYSApNPGQAAAAAAAPIAVFxFGV</td><td>Yes</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Motif 17</td><td>11</td><td>GPtGtQLxIVY</td><td>Yes</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Motif 18</td><td>15</td><td>FARGINxVRAETVRE</td><td>Yes</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr></tbody></table>

For each motif, the amino acid length (AA), the sequence and the presence/absence (Yes/–) in proteins of the PiT subfamilies (PHOSPHATE TRANSPORTER 2 (PHT2), bacterial PiT, metazoan PiT, fungal PiT, chlorophyte core PHOSPHATE TRANSPORTER B (core PTB) and streptophyte core PTB) are given. Upper case, lower case and ‘x’ indicate residues of the motifs conserved in 80%, 60% or < 60%, respectively, of the concerned proteins.

*Motif found in all core PTB proteins except *Dunaliella viridis* DvSPT1 and DvSPT2.
<figure><img src="image_4.png" /><figcaption>**Fig. 3** Complementation of the inorganic phosphate (Pi) uptake-deficient yeast mutant *ScΔpho2* by expression of *Marchantia polymorpha* core PHOSPHATE TRANSPORTER B (PTB)-encoding genes. The yeast strain Y2092 lacking the *ScPHO2* gene (*ScΔpho2*) was transformed with the *pCM189* vector expressing, under a tetracycline-repressible promoter, the coding sequence (CDS) of *MpPTB4*, *MpPTB6* or *MpPTB7*, the CDS of the yeast high-affinity Pi transporter *ScPHO84* or *ScPHO89* (positive controls), or the *pCM189* vector without insertion (wild-type (WT) Empty; negative control). The *PHO2* wild-type yeast strain (WT, Y178), isogenic to Y2092, was transformed with the *pCM189* empty vector and used as a positive control. To compare the growth of the different strains, 10 times serial dilutions of exponentially growing cells were spotted and grown for 11 d at 30 °C on medium containing the concentrations of Pi indicated and 10 mg l⁻¹ of doxycycline to repress the expression of the CDS, or no doxycycline.</figcaption></figure>

liverwort *M. polymorpha* grown in different concentrations of Pi. We found that the expression of the single *C. nitellarum* core PTB-encoding gene and of seven of the eight *M. polymorpha* core PTB-encoding genes was higher in low-Pi than in Pi-replete conditions (Fig. 4). Steady state levels of Cn*PTB* mRNAs were three-fold higher in low-Pi (0 mM) than in Pi-replete (1.6 mM) medium (Fig. 4a). In *M. polymorpha*, the steady state levels of the Mp*PTB2*, Mp*PTB3*, Mp*PTB4*, Mp*PTB5* and Mp*PTB7* mRNAs were between 12.5 times higher (Mp*PTB7*) and 730 times higher (Mp*PTB4*) in the rhizoids – the filamentous rooting cells of early diverging land plants – of plants grown in low-Pi than in Pi-replete conditions (Fig. 4b). In addition, Mp*PTB1* and Mp*PTB8* mRNAs were only detected in the rhizoids of plants grown in low-Pi conditions, indicating that they are exclusively expressed in low-Pi conditions (Fig. 4b). Only one *M. polymorpha* core *PTB* gene – Mp*PTB6* – was transcriptionally insensitive to low Pi in our conditions; the steady state levels of Mp*PTB6* mRNA were identical in low- and Pi-replete conditions (Fig. 4b). The increased expression of the only *C. nitellarum* and of the majority of the *M. polymorpha* core *PTB* genes – Cn*PTB*, Mp*PTB1*, Mp*PTB2*, Mp*PTB3*, Mp*PTB4*, Mp*PTB5*, Mp*PTB7* and Mp*PTB8* – in low-Pi growth conditions is typical of plasma membrane Pi transporter families catalysing Pi uptake from low-Pi environments (Mann *et al.*, 1989; Bun-Ya *et al.*, 1991; Versaw,

1995; Daram *et al.*, 1998; Martinez & Persson, 1998; Harris *et al.*, 2001; Rae *et al.*, 2003). The higher steady state mRNA levels of *M. polymorpha* Mp*PTB1*, Mp*PTB2*, Mp*PTB3*, Mp*PTB4*, Mp*PTB5*, Mp*PTB6* and Mp*PTB8* in rhizoids than in thallus support the hypothesis that these genes are involved in Pi uptake from the environment. Taken together, these data suggest that core *PTB* genes encode plasma membrane Pi transporters involved in Pi uptake in *C. nitellarum* and *M. polymorpha*.

Discussion

The uptake of Pi is necessary for plant growth and development, and here we report that members of the PTB family function as Pi transporters in streptophytes. PTB sequences have been identified previously in chlorophytes and hypothesized to be involved in Pi uptake on the basis of their sequence and structural similarity to the metazoan and fungal PiT Na⁺/Pi symporters (Chung *et al.*, 2003; Kobayashi *et al.*, 2003; Li *et al.*, 2006; Moseley *et al.*, 2006; Guan *et al.*, 2008; Djouani-Tahri *et al.*, 2011; Bergwitz *et al.*, 2012) and the ability of *D. viridis* core PTB proteins to increase the Na⁺ sensitivity of a yeast mutant defective in Na⁺ export (Li *et al.*, 2006). We demonstrate that core *PTB* genes from the liverwort *M. polymorpha* encode functional Pi transporters that complement a yeast mutant defective in high-affinity
<figure><img src="image_5.png" /><figcaption>**Fig. 4** Transcript level of core PHOSPHATE TRANSPORTER B (PTB)-encoding genes in *Marchantia polymorpha* and *Coleochaete nitellarum* grown in various inorganic phosphate (Pi) conditions. (a) Histograms indicating the mean transcript level (n = 3) of *CnPTB* in *C. nitellarum* under different Pi concentrations. Transcript levels were normalized with *CnEF1* expression. *The transcript level measured for each growth condition is significantly different from that measured in all other conditions (P < 0.05, Kruskal–Wallis test). Each biological replicate is represented by a circle, a square and a triangle for replicate 1, replicate 2 and replicate 3, respectively. (b) Histograms showing the mean transcript level (n = 3) of *MpPTB1*, *MpPTB2*, *MpPTB3*, *MpPTB4*, *MpPTB5*, *MpPTB6*, *MpPTB7* and *MpPTB8* in rhizoids (Rh) and thallus (Th) in low-Pi (LP) and high-Pi (HP) conditions in *M. polymorpha*. Transcript levels were normalized with the geometric mean of the *MpACT* and *MpSAND* expression levels. *The transcript levels measured in the conditions indicated by the ends of the brackets are significantly different from each other (P < 0.05, Kruskal–Wallis test). Each biological replicate is represented by a circle, a square and a triangle for replicate 1, replicate 2 and replicate 3, respectively.</figcaption></figure>

plasma membrane Pi transport. The presence of an N-terminal signal peptide on core PTB proteins suggests that core PTB Pi transporters are targeted to the secretory pathway and may be located at the plasma membrane. This hypothesis is supported by the location of the core PTB protein, MpPTB6, at the plasma membrane of *M. polymorpha*. Furthermore, the observation of higher steady state levels of most core *PTB* genes of *M. polymorpha* and the streptophyte alga *C. nitellarum* in low-Pi than in Pi-replete growth conditions, and of most core *PTB* genes of *M. polymorpha* in rhizoids than in thallus, resembles the expression of members of the PHT1 family (Mudge *et al.*, 2002; Nussaume *et al.*, 2011; Qin *et al.*, 2012) and is characteristic of

Pi-responsive genes, including genes encoding Pi transporters involved in the uptake of Pi from low-Pi environments (Mann *et al.*, 1989; Bun-Ya *et al.*, 1991; Versaw, 1995; Daram *et al.*, 1998; Martinez & Persson, 1998; Harris *et al.*, 2001; Rae *et al.*, 2003). Taken together, these data are consistent with core PTB proteins being plasma membrane Pi transporters in strepto-phytes.

In angiosperms, proteins in the PHT1 family catalyse the uptake of Pi from the environment through H⁺/Pi symport (Leggewie *et al.*, 1997; Mitsukawa *et al.*, 1997; Daram *et al.*, 1998; Rae *et al.*, 2003; Liu *et al.*, 2008; Nussaume *et al.*, 2011). Although members of the PHT1 Pi transporter family have been
reported in streptophyte algae and early diverging land plants, their function has not been demonstrated in these taxa (Wang et al., 2008; Delaux et al., 2015; Saint-Marcoux et al., 2015). However, the higher steady state level of one M. polymorpha and two P. patens PHT1 mRNAs in plants grown on low-Pi relative to Pi-replete conditions (Wang et al., 2008; Saint-Marcoux et al., 2015) is consistent with the hypothesis that PHT1 proteins are active in Pi uptake in early diverging land plants. The presence of functional core PTB Pi transporters in early diverging land plants and streptophyte algae suggests that two families of plasma membrane Pi transporters – core PTB and PHT1 – act alongside each other in Pi uptake in these species. Such a dual plasma membrane Pi transporter system has not been described previously in streptophytes. Two high-affinity Pi uptake systems act in fungi; H⁺/Pi transporters homologous to PHT1 proteins and Na⁺/Pi transporters homologous to PTB proteins are active in Pi uptake in low external Pi conditions in N. crassa, S. cerevisiae and Yarrowia lipolytica. Differences in pH optima and counter-ion selectivity of the fungal Na⁺/Pi and H⁺/Pi symporters have led to the hypothesis that different transporters are active in different environmental conditions, providing the organisms with a wide adaptive range (Versaw, 1995; Zvyagilskaya & Persson, 2003; Dick et al., 2014). This could also be true in streptophyte algae and early diverging land plants, where both PHT1 and core PTB proteins are probably active in Pi uptake.

H⁺/Pi symport by PHT1 proteins in angiosperms depends on the generation of an H⁺ gradient through the activity of the plasma membrane H⁺-ATPase (Leggewie et al., 1997; Mitsukawa et al., 1997; Daram et al., 1998; Rae et al., 2003; Liu et al., 2008; Nussaume et al., 2011). Such an H⁺ gradient cannot be generated in an aquatic environment because of the rapid dispersion and reaction of H⁺ with hydroxide ions in the water (Agmon et al., 2016). Moreover, both marine and fresh water habitats occupied by charophyte and chlorophyte algae are alkaline (Raven, 1984; Walker & Sanders, 1991; Rubio et al., 2005; Moseley et al., 2006; Chan et al., 2012). The hypothesis that Na⁺ could be used as a driving force by organisms occupying environments in which the production of an H⁺ gradient is not possible, such as an alkaline or aquatic environment, was originally proposed after physiological studies showed that the uptake of several nutrients, including Pi, amino acids and glucose, was dependent on Na⁺ uptake in algae, but on H⁺ uptake in terrestrial angiosperms (Ullrich-Eberius & Yinchol, 1974; Raven, 1984; Walker & Sanders, 1991; Bush, 1993; Reid et al., 2000; Weiss et al., 2001; Rubio et al., 2005; Kuzyakov & Jones, 2006; Svennerstam et al., 2011; Chan et al., 2012). Consistent with this model is the observation that *D. viridis* core PTB proteins catalyse Na⁺ transport, suggesting that these proteins are probably Na⁺/Pi symporters in aquatic chlorophytes (*Li* et al., 2006). Chlorophytes, streptophyte algae, liverworts, mosses and hornworts require moist habitats. Thus, the activity of core PTB in these lineages could facilitate Na⁺/Pi uptake from water. Our report that proteins involved in Na⁺- and H⁺-dependent Pi uptake co-exist in streptophyte algae and early diverging land plants suggests that Na⁺ and H⁺ symport systems co-existed in streptophytes before plants colonized the land. Early diverging land plants inherited the dual Pi uptake system

comprising both core PTB Na⁺/Pi and PHT1 H⁺/Pi symporters from streptophyte algae, where it is conserved in extant liverworts, mosses and hornworts, whose life cycle depends on the presence of free water.

There are PTB proteins in tracheophytes (lyophytes, monilophytes, gymnosperms and angiosperms). These tracheophyte proteins belong to PTB clade 4, a PTB clade that is phylogenetically distinct from the core PTB proteins (Figs 2, S2, S3). During tracheophyte evolution, members of PTB clade 4 lost the N-terminal signal peptide (Table S3), the GANDVAN signature domains and the signature glutamates that are required for Pi transport in metazoan and fungal PiT proteins (Bøttger & Pedersen, 2002, 2005; Bøttger et al., 2006; Andersson et al., 2012) (Table S4; Fig. S6). This suggests that PTB clade 4 proteins are not Pi transporters and are not located at the plasma membrane. Therefore, the absence of core PTB proteins in tracheophytes suggests that land plants lost the core PTB Na⁺/Pi transport function as plants radiated into drier habitats, leading to the situation in angiosperms in which PHT1 is the major Pi uptake system (Nussaume et al., 2011; Ayadi et al., 2015). These data suggest that there was a gradual transition from a dual H⁺/Pi and Na⁺/Pi uptake system in early diverging land plants to a Pi uptake system entirely dependent on H⁺/Pi uptake in late diverging groups during the course of land plant evolution as they adapted to drier habitats. PTB proteins are not encoded in the genome of *Z. marina* (Olsen et al., 2016) despite physiological evidence for Na⁺/Pi uptake (Rubio et al., 2005). This suggests that a PTB-independent Na⁺/Pi uptake mechanism evolved secondarily in angiosperms that re-adapted to aquatic habitats.

## Acknowledgements

This research was supported by a European Research Council Advanced award (EVO500 project no. 25028) and a Marie Skłodowska-Curie award (PLANTORIGINS project no. 238640) to L.D. A.J.H. was supported by a Biotechnology and Biological Sciences Research Council Doctoral Training Partnership Scholarship (Grant BB/J014427/1). We are grateful to Professor D. Domozych (Skidmore College, NY, USA) for providing *C. nitellarum*, to Professor T. Kohchi and Dr K. Ishizaki for providing *M. polymorpha*, to Dr T. Ueda for the *pMpSYP12A:mCitrine-MpSYP12A:Term* construct and to Dr I. Moore for the *p35S:YFP:HDEL:Term* construct. Thanks are due to Dr R. Toth and Dr S. Honkanen for helpful discussions.

## Author contributions

C.B. designed the research. C.B. and L.D. wrote the paper. C.B., F.P.L.C and A.L-V. identified the plant PiT sequences. H.P. performed the expression analysis in *M. polymorpha*. C.B., H.P. and B.P. performed the yeast complementation test. C.B. and H.B. produced the RNA sample for *C. nitellarum* RNAseq. C.C., A.J.H. and S.K. performed the transcriptome assembly. C.B. and C.C. captured and analysed the confocal images. C.B. performed all other experiments and analysed all the data.
References

Abascal F, Zardoya R, Posada D. 2005. ProTTest: selection of best-fit models of protein evolution. *Bioinformatics* 21: 2104–2105.

Agmon N, Bakker HJ, Campen RK, Henchman RH, Pohl P, Roke S, Thämer M, Hassanali A. 2016. Protons and hydroxide ions in aqueous systems. *Chemical Reviews* 116: 7642–7672.

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. *Journal of Molecular Biology* 215: 403–410.

Andersson M, Samyn D, Persson B. 2012. Mutational analysis of conserved glutamic acids of Pho89, a *Saccharomyces cerevisiae* high-affinity inorganic phosphate:Na⁺ symporter. *Biologia* 67: 1056–1061.

Ayadi A, David P, Arrighi J-F, Chiarenza S, Thibaud M-C, Nussaume L, Marin E. 2015. Reducing the genetic redundancy of Arabidopsis PHOSPHATE TRANSPORTER1 transporters to study phosphate uptake and signaling. *Plant Physiology* 167: 1511–1526.

Bailey TL, Elkan C. 1994. Fitting a mixture model by expectation maximization to discover motifs in bipolymers. In: *Proceedings of the second international Conference on Intelligent systems for molecular biology*. Menlo Park, CA, USA: AAAI Press, 28–36.

Bergwitz C, Rasmussen MD, DeRobertis C, Wee MJ, Sinha S, Chen HH, Huang J, Perrimon N. 2012. Roles of major facilitator superfamily transporters in phosphate response in Drosophila. *PLoS ONE* 7: e31730.

Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffolding pre-assembled contigs using SSPACE. *Bioinformatics* 27: 578–579.

Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. *Bioinformatics* 30: 2114–2120.

Bötger P, Hede SE, Grunnet M, Høyer B, Klærke DA, Pedersen L. 2006. Characterization of transport mechanisms and determinants critical for Na⁺-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2. *American Journal of Physiology-Cell Physiology* 291: C1377–C1387.

Bötger P, Pedersen L. 2002. Two highly conserved glutamate residues critical for type III sodium-dependent phosphate transport revealed by uncoupling transport function from retroviral receptor function. *Journal of Biological Chemistry* 277: 42741–42747.

Bötger P, Pedersen L. 2005. Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium-dependent phosphate transport function of human PiT2. *FEBS Journal* 272: 3060–3074.

Breuninger H, Thamm A, Streubel S, Sakayama H, Nishiyama T, Dolan L. 2016. Diversification of a transcription factor family led to the evolution of antagonistically acting genetic regulators of root hair growth. *Current Biology* 26: 1622–1628.

Bucher M. 2006. Functional biology of plant phosphate uptake at root and mycorrhiza interfaces. *New Phytologist* 173: 11–26.

Bun-Ya M, Nishimura M, Harashima S, Oshima Y. 1991. The PH084 gene of *Saccharomyces cerevisiae* encodes an inorganic phosphate transporter. *Molecular and Cellular Biology* 11: 3229–3238.

Bush DR. 1993. Proton-coupled sugar and amino acid transporters in plants. *Annual Review of Plant Biology* 44: 513–542.

Butler J, MacCallum I, Kleber M, Shlyakhter IA, Belmonte MK, Lander ES, Nusbaum C, Jaffe DB. 2008. ALLPATHS: de novo assembly of whole-genome shotgun microreads. *Genome Research* 18: 810–820.

Chan CX, Zäuner S, Wheeler G, Grossman AR, Prochnik SE, Blouin NA, Zhuang Y, Benning C, Berg GM, Yarish C. 2012. Analysis of Porphyra membrane transporters demonstrates gene transfer among photosynthetic eukaryotes and numerous sodium-coupled transport systems. *Plant Physiology* 158: 2001–2012.

Chen A, Hu J, Sun S, Xu G. 2007. Conservation and divergence of both phosphate and mycorrhiza regulated physiological responses and expression patterns of phosphate transporters in solanaceous species. *New Phytologist* 173: 817–831.

Chen DC, Yang BC, Kuo TT. 1992. One-step transformation of yeast in stationary phase. *Current Genetics* 21: 83–84.

Chrispeels MJ, Raikhel NV. 1992. Short peptide domains target proteins to the plant vacuoles. *Cell* 68: 613–616.

Chung C-C, Hwang S-PL, Chang J. 2003. Identification of a high-affinity phosphate transporter gene in a prasinophyte alga, *Tetraselmis chui*, and its expression under nutrient limitation. *Applied and Environmental Microbiology* 69: 754–759.

Corpet F. 1988. Multiple sequence alignment with hierarchical clustering. *Nucleic Acids Research* 16: 10881–10890.

Crusoe MR, Alameldin HF, Awad S, Boucher E, Caldwell A, Cartwright R, Charbonneau A, Constantinides B, Edvenson G, Fay S. 2015. The khmer software package: enabling efficient nucleotide sequence analysis. *F1000Research* 4: 900.

Daram P, Brunner S, Persson BL, Amrhein N, Bucher M. 1998. Functional analysis and cell-specific expression of a phosphate transporter from tomato. *Planta* 206: 225–233.

Delaux P-M, Radhakrishnan GV, Jayaraman D, Cheema J, Malbreil M, Volkening JD, Sekimoto H, Nishiyama T, Melkonian M, Pokorny L *et al.* 2015. Algal ancestor of land plants was preadapted for symbiosis. *Proceedings of the National Academy of Sciences, USA* 112: 13390–13395.

Dick CF, Dos-Santos AL, Meyer-Fernandes JR. 2014. Inorganic phosphate uptake in unicellular eukaryotes. *Biochimica et Biophysica Acta (BBA)—General Subjects* 1840: 2123–2127.

Djouani-Tahri EB, Sanchez F, Lozano J-C, Bouget F-Y. 2011. A phosphate-regulated promoter for fine-tuned and reversible overexpression in *Ostreococcus* application to circadian clock functional analysis. *PLoS ONE* 6: e28471.

Emanuelsson O, Brunak S, von Heijne G, Nielsen H. 2007. Locating proteins in the cell using TargetP, SignalP and related tools. *Nature Protocols* 2: 953–971.

Gari E, Piedrafita L, Aldea M, Herrero E. 1997. A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in *Saccharomyces cerevisiae*. *Yeast* 13: 837–848.

Guan Z, Meng X, Sun Z, Xu Z, Song R. 2008. Characterization of duplicated *Dunaliella viridis* *SPT1* genes provides insights into early gene divergence after duplication. *Gene* 423: 36–42.

Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. *Systematic Biology* 59: 307–321.

Harris RM, Webb DC, Howitt SM, Cox GB. 2001. Characterization of PitA and PitB from *Escherichia coli*. *Journal of Bacteriology* 183: 5008–5014.

Harrison MJ, Dewbre GR, Liu J. 2002. A phosphate transporter from *Medicago truncatula* involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi. *Plant Cell* 14: 2413–2429.

Hofmann K, Stoffel W. 1993. TMbase—A database of membrane spanning protein segments. *Biological Chemistry Hoppe-Seyler* 374: 166.

Huang X, Madan A. 1999. CAP3: a DNA sequence assembly program. *Genome Research* 9: 868–877.

Ishizaki K, Chiyoda S, Yamato KT, Kohchi T. 2008. Agrobacterium-mediated transformation of the haploid liverwort *Marchantia polymorpha* L., an emerging model for plant biology. *Plant and Cell Physiology* 49: 1084–1091.

Jiang W, Chen S-Y, Wang H, Li D-Z, Wiens JJ. 2014. Should genes with missing data be excluded from phylogenetic analyses? *Molecular Phylogenetics and Evolution* 80: 308–318.

Johnson CM, Stout PR, Broyer TC, Carlton AB. 1957. Comparative chlorine requirements of different plant species. *Plant and Soil* 8: 337–353.

Kanazawa T, Era A, Minamino N, Shikano Y, Fujimoto M, Uemura T, Nishihama R, Yamato KT, Ishizaki K, Nishiyama T *et al.* 2016. SNARE molecules in *Marchantia polymorpha*: unique and conserved features of the membrane fusion machinery. *Plant and Cell Physiology* 57: 307–324.

Katoh K, Kuma K, Toh H, Miyata T. 2005. MAFFT version 5: improvement in accuracy of multiple sequence alignment. *Nucleic Acids Research* 33: 511–518.

Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. *Molecular Biology and Evolution* 30: 772–780.

Kobayashi I, Fujiwara S, Shimogawara K, Kaise T, Usuda H, Tsuzuki M. 2003. Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on Chlamydomonas. *Plant and Cell Physiology* 44: 597–606.

Kopylova E, Noël L, Touzet H. 2012. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. *Bioinformatics* 28: 3211–3217.
Kuzyakov Y, Jones DL. 2006. Glucose uptake by maize roots and its transformation in the rhizosphere. *Soil Biology and Biochemistry* **38**: 851–860.

Le SQ, Gascuel O. 2008. An improved general amino acid replacement matrix. *Molecular Biology and Evolution* **25**: 1307–1320.

Leggewie G, Willmitzer L, Riesmeier JW. 1997. Two cDNAs from potato are able to complement a phosphate uptake-deficient yeast mutant: identification of phosphate transporters from higher plants. *Plant Cell* **9**: 381–392.

Li Q, Gao X, Sun Y, Zhang Q, Song R, Xu Z. 2006. Isolation and characterization of a sodium-dependent phosphate transporter gene in *Dunaliella viridis*. *Biochemical and Biophysical Research Communications* **340**: 95–104.

Li S-H, Xia B-B, Zhang C, Cao J, Bai L-H. 2012. Cloning and characterization of a phosphate transporter gene in *Dunaliella salina*. *Journal of Basic Microbiology* **52**: 429–436.

Liu F, Chang X-J, Ye Y, Xie W-B, Wu P, Lian X-M. 2011. Comprehensive sequence and whole-life-cycle expression profile analysis of the phosphate transporter gene family in rice. *Molecular Plant* **4**: 1105–1122.

Liu J, Versaw WK, Pumplin N, Gomez SK, Blaylock LA, Harrison MJ. 2008. Closely related members of the *Medicago truncatula* PHT1 phosphate transporter gene family encode phosphate transporters with distinct biochemical activities. *Journal of Biological Chemistry* **283**: 24673–24681.

Mann BJ, Bowman BJ, Grotelueschen J, Metzenberg RL. 1989. Nucleotide sequence of *pho-4*, encoding a phosphate-repressible phosphate permease of *Neurospora crassa*. *Gene* **83**: 281–289.

Martinez P, Persson BL. 1998. Identification, cloning and characterization of a derepressible Na⁺-coupled phosphate transporter in *Saccharomyces cerevisiae*. *Molecular and General Genetics* **258**: 628–638.

Mimura T, Reid RJ, Ohsumi Y, Smith FA. 2002. Induction of the Na⁺/Pi cotransport system in the plasma membrane of *Chara corallina* requires external Na⁺ and low levels of Pi. *Plant, Cell & Environment* **25**: 1475–1481.

Mitsukawa N, Okumura S, Shirano Y, Sato S, Kato T, Harashima S, Shibata D. 1997. Overexpression of an *Arabidopsis thaliana* high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions. *Proceedings of the National Academy of Sciences, USA* **94**: 7098–7102.

Moseley JL, Chang CW, Grossman AR. 2006. Genome-based approaches to understanding phosphorus deprivation responses and PSR1 control in *Chlamydomonas reinhardtii*. *Eukaryotic Cell* **5**: 26–44.

Mudge SR, Rae AL, Diatloff E, Smith FW. 2002. Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis. *Plant Journal* **31**: 341–353.

Nichols HW. 1973. Growth media-freshwater. In: Stein JR, ed. *Handbook of phycological methods: culture methods and growth measurements*. Cambridge, UK: Cambridge University Press, 39–78.

Nikolenko SI, Korobeynikov AI, Alekseyev MA. 2013. BayesHammer: Bayesian clustering for error correction in single-cell sequencing. *BMC Genomics* **14**: 1.

Nussaume L, Kanno S, Javot H, Marin E, Pochon N, Ayadi A, Nakanishi TM, Thibaud M-C. 2011. Phosphate import in plants: focus on the PHT1 transporters. *Frontiers in Plant Science* **2**: 235–246.

O'Hara B, Johann SV, Klinger HP, Blair DG, Rubinson H, Dunn KJ, Sass P, Vitek SM, Robins T. 1990. Characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus. *Cell Growth & Differentiation* **1**: 119–127.

Olsen JL, Rouzé P, Verhelst B, Lin Y-C, Bayer T, Collen J, Dattolo E, De Paoli E, Dittami S, Maumus F. 2016. The genome of the seagrass *Zostera marina* reveals angiosperm adaptation to the sea. *Nature* **530**: 331–335.

Pinson B, Merle M, Franconi J-M, Daignan-Fornier B. 2004. Low affinity orthophosphate carriers regulate PHO gene expression independently of internal orthophosphate concentration in *Saccharomyces cerevisiae*. *Journal of Biological Chemistry* **279**: 35273–35280.

Pinson B, Vaur S, Sagot I, Coulpier F, Lemoine S, Daignan-Fornier B. 2009. Metabolic intermediates selectively stimulate transcription factor interaction and modulate phosphate and purine pathways. *Genes & Development* **23**: 1399–1407.

Proust H, Honkanen S, Jones VAS, Morieri G, Prescott H, Kelly S, Ishizaki K, Kohchi T, Dolan L. 2016. RSL class I genes controlled the development of

epidermal structures in the common ancestor of land plants. *Current Biology* **26**: 93–99.

Qin L, Guo Y, Chen L, Liang R, Gu M, Xu G, Zhao J, Walk T, Liao H. 2012. Functional characterization of 14 Pht1 family genes in yeast and their expressions in response to nutrient starvation in soybean. *PLoS ONE* **7**: e47726.

Rae AL, Cybinski DH, Jarmey JM, Smith FW. 2003. Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among members of the Pht1 family. *Plant Molecular Biology* **53**: 27–36.

Raven JA. 1984. *Energetics and transport in aquatic plants*. New York, NY, USA: AR Liss.

Reid RJ, Mimura T, Ohsumi Y, Walker NA, Smith FA. 2000. Phosphate uptake in *Chara*: membrane transport via Na⁺/Pi cotransport. *Plant, Cell & Environment* **23**: 223–228.

Rubio L, Linares-Rueda A, García-Sánchez MJ, Fernández JA. 2005. Physiological evidence for a sodium-dependent high-affinity phosphate and nitrate transport at the plasma membrane of leaf and root cells of *Zostera marina* L. *Journal of Experimental Botany* **56**: 613–622.

Ruhfel BR, Gitzendanner MA, Soltis PS, Soltis DE, Burleigh JG. 2014. From algae to angiosperms—in-ferring the phylogeny of green plants (Viridiplantae) from 360 plastid genomes. *BMC Evolutionary Biology* **14**: 23.

Saint-Marcoux D, Proust H, Dolan L, Langdale JA. 2015. Identification of reference genes for real-time quantitative PCR experiments in the liverwort *Chantlandia polymorpha*. *PLoS ONE* **10**: e0118678.

Salaün C, Maréchal V, Heard JM. 2004. Transport-deficient Pit2 phosphate transporters still modify cell surface oligomers structure in response to inorganic phosphate. *Journal of Molecular Biology* **340**: 39–47.

Salaün C, Rodrigues P, Heard JM. 2001. Transmembrane topology of Pit2, a phosphate transporter-retrovirus receptor. *Journal of Virology* **75**: 5584–5592.

Simpson JT, Durbin R. 2012. Efficient *de novo* assembly of large genomes using compressed data structures. *Genome Research* **22**: 549–556.

Solovyev VV. 2007. Statistical approaches in Eukaryotic gene prediction. In: Balding D, Cannings C, Bishop M, eds. *Handbook of statistical genetics*. Hoboken, NJ, USA: Wiley-Interscience, 97–159.

Svennerstam H, jämtgard S, Ahmad I, Huss-Danell K, Näsholm T, Ganeteg U. 2011. Transporters in Arabidopsis roots mediating uptake of amino acids at naturally occurring concentrations. *New Phytologist* **191**: 459–467.

Teh O, Moore I. 2007. An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells. *Nature* **448**: 493–496.

Tyra HM, Linka M, Weber AP, Bhattacharya D. 2007. Host origin of plastid solute transporters in the first photosynthetic eukaryotes. *Genome Biology* **8**: R212.

Ullrich-Eberius CI, Yinchol Y. 1974. Phosphate uptake and its pH-dependence in halophytic and glycophytic algae and higher plants. *Oecologia* **17**: 17–26.

Van Zeijl M, Johann SV, Closs E, Cunningham J, Eddy R, Shows TB, O’Hara B. 1994. A human amphotropic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family. *Proceedings of the National Academy of Sciences, USA* **91**: 1168–1172.

Versaw WK. 1995. A phosphate-repressible, high-affinity phosphate permease is encoded by the *pho-5* gene of *Neurospora crassa*. *Gene* **153**: 135–139.

Versaw WK, Harrison MJ. 2002. A chloroplast phosphate transporter, PHT2;1, influences allocation of phosphate within the plant and phosphate-starvation responses. *Plant Cell* **14**: 1751–1766.

Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. *PLoS ONE* **9**: e112963.

Walker NA, Sanders D. 1991. Sodium-coupled solute transport in charophyte algae: a general mechanism for transport energization in plant cells? *Planta* **185**: 443–445.

Wang Y, Secco D, Poirier Y. 2008. Characterization of the *PHO1* gene family and the responses to phosphate deficiency of *Physcomitrella patens*. *Plant Physiology* **146**: 646–656.
Weiss M, Haimovich G, Pick U. 2001. Phosphate and sulfate uptake in the halotolerant alga *Dunaliella* are driven by Na⁺-symport mechanism. *Journal of Plant Physiology* **158**: 1519–1525.

Werner A, Dehmelt L, Nalbant P. 1998. Na⁺-dependent phosphate cotransporters: the NaPi protein families. *Journal of Experimental Biology* **201**: 3135–3142.

Werner A, Kinne RK. 2001. Evolution of the Na-Pi cotransport systems. *American Journal of Physiology–Regulatory, Integrative and Comparative Physiology* **280**: R301–R312.

Zhao L, Versaw WK, Liu J, Harrison MJ. 2003. A phosphate transporter from *Medicago truncatula* is expressed in the photosynthetic tissues of the plant and located in the chloroplast envelope. *New Phytologist* **157**: 291–302.

Zvyagilskaya R, Persson BL. 2003. Dual regulation of proton- and sodium-coupled phosphate transport systems in the Yarrowia lipolytica yeast by extracellular phosphate and pH. *IUBMB Life* **55**: 151–154.

## Supporting Information

Additional Supporting Information may be found online in the Supporting Information tab for this article:

**Fig. S1** Multiple amino acid alignment sequences used for the Pi TRANSPORTER (PiT) phylogeny of green plants.

**Fig. S2** Multiple amino acid alignment sequences used for the enlarged Pi TRANSPORTER (PiT) phylogeny of green plants.

**Fig. S3** Maximum-likelihood tree of the Pi TRANSPORTER (PiT) sequences of green plants.

**Fig. S4** Maximum-likelihood tree of the enlarged set of Pi TRANSPORTER (PiT) sequences of green plants.

**Fig. S5** The MpPTB6-YFP fusion protein is located at the plasma membrane and endoplasmic reticulum.

**Fig. S6** Location of conserved motifs in the Pi TRANSPORTER (PiT) amino acid sequences.

**Table S1** Cross-reference information for the *PiT* sequences used for the Pi TRANSPORTER (PiT) phylogeny of green plants

**Table S2** List of primers for cloning, vector construction and expression analysis

**Table S3** TARGETP prediction of subcellular targeting of PHOSPHATE TRANSPORTER B (PTB) clade 4 proteins

**Table S4** Conserved motifs of Pi TRANSPORTER (PiT) sub-families identified by MEME (Multiple Em for Motif Elicitation) in PHOSPHATE TRANSPORTER B (PTB) clade 4

Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the *New Phytologist* Central Office.

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