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First published online April 30, 2004; 10.1104/pp.103.037945 Plant Physiology 135:400-411 (2004) © 2004 American Society of Plant Biologists Structure and Expression Profile of the Arabidopsis PHO1 Gene Family Indicates a Broad Role in Inorganic Phosphate Homeostasis1,[w]Département de Biologie Moléculaire Végétale, Bâtiment de Biologie, Université de Lausanne, CH–1015 Lausanne, Switzerland
PHO1 has been recently identified as a protein involved in the loading of inorganic phosphate into the xylem of roots in Arabidopsis. The genome of Arabidopsis contains 11 members of the PHO1 gene family. The cDNAs of all PHO1 homologs have been cloned and sequenced. All proteins have the same topology and harbor a SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the C-terminal hydrophobic portion. The SPX and EXS domains have been identified in yeast (Saccharomyces cerevisiae) proteins involved in either phosphate transport or sensing or in sorting proteins to endomembranes. The Arabidopsis genome contains additional proteins of unknown function containing either a SPX or an EXS domain. Phylogenetic analysis indicated that the PHO1 family is subdivided into at least three clusters. Reverse transcription-PCR revealed a broad pattern of expression in leaves, roots, stems, and flowers for most genes, although two genes are expressed exclusively in flowers. Analysis of the activity of the promoter of all PHO1 homologs using promoter-  -glucuronidase fusions revealed a predominant expression in the vascular tissues of roots, leaves, stems, or flowers. -Glucuronidase expression is also detected for several promoters in nonvascular tissue, including hydathodes, trichomes, root tip, root cortical/epidermal cells, and pollen grains. The expression pattern of PHO1 homologs indicates a likely role of the PHO1 proteins not only in the transfer of phosphate to the vascular cylinder of various tissues but also in the acquisition of phosphate into cells, such as pollen or root epidermal/cortical cells.
Phosphorus (P) is an essential macronutrient for all living organisms. It serves various basic biological functions as a structural element in nucleic acids and phospholipids, in energy metabolism, in the activation of metabolic intermediates, as a component in signal transduction cascades, and in the regulation of enzymes.
Of the major nutrients, P is the most dilute and the least mobile in soil. P is absorbed by plants as orthophosphate (inorganic phosphate [Pi]). Pi concentration in the soil solution hardly reaches 10 µM and may even drop to submicromolar levels at the root/soil interface. Plants have evolved a series of metabolic and developmental adaptations aimed at increasing the acquisition of this vital but poorly available nutrient from the soil, as well as to sustain plant growth and survival under low P availability (for review, see Poirier and Bucher, 2002 Transport of Pi is a dynamic process that occurs across various membranes, between cells and tissues, as well as between organelles. For example, once imported into the root epidermal or cortical cells, Pi must be exported into the xylem vessels of the root for its transfer, via the transpiration stream, to the shoot, where Pi is imported again into leaf cells. Under Pi deficiency, Pi is redistributed from the old, fully expanded source leaves toward young, expending sink leaves, a process requiring Pi export to the phloem vessel. Pi deficiency is also known to increase the Pi import capacity of cells through, in part, increase in the level of expression of Pi transporters. Transport of Pi across the tonoplast is an important process in Pi homeostasis. In plants grown in Pi-sufficient conditions, a major portion of Pi is transported into the vacuole, while under Pi-deficient conditions, the Pi is exported out of the vacuole in order to maintain a relatively stable cytoplasmic Pi concentration.
Our knowledge of Pi transport and homeostasis in higher plants has been extended in recent years by the cloning of several genes encoding H+-Pi cotransporters involved in Pi uptake into cells (Raghothama, 1999
A gene homologous to the animal Na-Pi cotransporter gene has been isolated in Arabidopsis that encodes a low-affinity H+-Pi cotransporter expressed preferentially in leaves (Pht2;1; Daram et al., 1999
The pho1 mutant of Arabidopsis is deficient in loading root Pi into the xylem vessels, resulting in strong Pi deficiency in all the above-ground tissues (Poirier et al., 1991
Cloning and Sequencing of the PHO1 Gene Family Analysis of the Arabidopsis genome with the PHO1 protein sequence revealed the presence of 10 homologous genes (identified as PHO1;H1 to PHO1;H10, hereafter referred as PHO1 homologs H1 to H10; Table I). Expressed sequence tags (ESTs) or full-length cDNA have only been reported for two of the potential 10 homologs, indicating that this gene family was underrepresented in the various collections of expressed genes. Based on the structure of the PHO1 gene as well as the sequence of cDNAs and ESTs for the homologs H1, H2, and H5 genes, primers have been designed to amplify and clone by reverse transcription (RT)-PCR the full coding sequence of all PHO1 homologs. Initial cloning of cDNAs in Escherichia coli using the pBluescript KS+ vector gave rise only to clones having insertions or rearrangements affecting the open reading frame. The cDNAs fragments obtained by RT-PCR have thus been ligated into the yeast-E. coli shuttle vector pYES2 and the ligation product transformed directly into yeast. Positive clones were identified by PCR and sequenced. The sequence of the H1 clone obtained by this approach was found to be identical to a full-length cDNA reported in GenBank (R14567). Out of the nine PHO1 homologs, for which only a predicted sequence was found on the Arabidopsis Information Resource (TAIR; http://www.arabidopsis.org) database, the cDNA sequences of six genes were found to be different from the TAIR prediction. In all cases, the changes involved splicing sites and modified the predicted sequences of the proteins over short stretches. The sequences of all PHO1 homologs have been submitted to GenBank.
Pairwise comparison of the PHO1 protein family revealed levels of identity or similarity ranging from 29% amino acid identity and 49% amino acid similarity between PHO1 and the H6 homolog to 86% amino acid identity and 94% amino acid similarity between the homologs H7 and H8 (Fig. 1). Analysis of the hydropathy profiles of all PHO1 homologs revealed a pattern that was very similar to PHO1, with the first half of the protein being hydrophilic and the second half being mostly hydrophobic and containing at least six potential transmembrane-spanning domains (Supplemental Fig. 1). Alignment of the proteins revealed extensive areas of similarity between the PHO1 protein family members (Fig. 2). Within the hydrophilic half of the proteins, three conserved domains are found separated by two areas of low similarity. By contrast, extensive similarities are found throughout the whole hydrophobic half of the proteins.
Two distinct full-length cDNAs encoding proteins having homology to PHO1 have been reported for rice (O. sativa cv japonica). Clone J023079I02 shows highest similarity to PHO1, with 49% amino acid identity and 62% amino acid similarity, while clone J013095I12 shows highest similarity to the H1 homolog with 55% amino acid identity and 66% amino acid similarity. The hydropathy profile, as well as the overall domain structure, are conserved between the rice and the Arabidopsis PHO1 genes (Fig. 2; Supplemental Fig. 1). An unrooted phylogenetic tree constructed from the Arabidopsis and rice sequences revealed the presence of three clusters (Fig. 3). One cluster regroups the two rice proteins with the Arabidopsis PHO1 and the H1 homolog, a second group includes the homologs H2, H3, H5, and H6, while a third group includes the homologs H4, H7, and H8. The H9 and H10 homologs do not apparently cluster with any other PHO1 proteins. The observed clustering is the result of amino acid sequence differences spread throughout the whole proteins and not limited to a specific region.
Structure and Distribution of PHO1 Genes in the Genome
More than 60% of the Arabidopsis genome is represented within duplicated regions (The Arabidopsis Genome Initiative, 2000
Analysis of the intron/exon structure of the PHO1 genes family revealed a greater divergence for regions encoding the N-terminal hydrophilic domains compared to the C-terminal hydrophobic domains (Fig. 5). Thus, the intron/exon structure of the C-terminal hydrophobic domains typically includes seven to eight introns, with the position and size of introns being fairly well conserved between homologs, with the exception of H6, which has distinctively larger introns. By contrast, the intron/exon structure of the N-terminal hydrophilic domains is more variable, with intron number ranging between three to six and the size of the first intron ranging from approximately 80 to 1,060 bp. Thus, the greater divergence within the N-terminal hydrophilic domains of the PHO1 protein family observed at the amino acid level is also extended at the gene structure level.
The PHO1 Proteins Harbor the SPX and EXS Domains The PHO1 proteins show weak but significant similarities to a number of proteins found in Arabidopsis as well as in nonplant eukaryotes. These similarities are mainly confined to two domains, named SPX and EXS.
The SPX domain was named after the proteins SYG1 and PHO81 of yeast and XPR1 of human (PFAM entry PF03105; www.sanger.ac.uk). The SPX domain can typically be subdivided into three smaller domains of 35 to 47 amino acids. In most proteins, such as SYG1 and PHO81, the three subdomains are found close together, typically within a stretch of 180 amino acids. Analysis of the N-terminal hydrophilic half of the PHO1 proteins revealed the presence of the SPX domain in all proteins, with the three subdomains being found within a stretch of 300 to 360 amino acids and being separated from each other by larger regions of low similarity (Fig. 2). Yeast has at least eight proteins having a SPX domain. These include SYG1, which has been initially identified as a suppressor of the mating pheromone signal; the VTC1, VTC2, VTC3, and VTC4 proteins involved in sorting proteins to the vacuolar membrane; and the PHO81, PHO87, PHO90, and PHO91 involved in phosphate sensing and transport (Spain et al., 1995
The EXS domain was named after the yeast ERD1, involved in the localization of endogenous endoplasmic reticulum proteins, the human XPR1, and the yeast SYG1 (PFAM entry PF03124; www.sanger.ac.uk). In contrast to the SYG1 and XPR1 proteins, which have both a SPX and EXS domain, the majority of proteins in yeast having one of these domains do not harbor the other. In Arabidopsis, only the members of the PHO1 proteins family possess both a SPX and EXS domain. In addition to all PHO1 homologs, two proteins in Arabidopsis have an EXS domain (Fig. 7). At5g35730 and At2g32295 are proteins of unknown function having several predicted transmembrane domains. At5g35730 is predicted to be localized to the chloroplast, while At2g32295 is predicted to be mitochondrial.
Expression Profile of the PHO1 Gene Family
The expression profile of the PHO1 gene family was first examined by RT-PCR using primers that were specific to each PHO1 homolog (Fig. 8). Tissues were harvested either from plants grown in fertilized soil (leaves, stems, and flowers) or from roots growing in liquid cultures. The majority of the PHO1 homologs (H1, H2, H3, H4, H5, H7, H8, and H10) were found expressed at various levels in roots, leaves, stems, and flowers. However, the PHO1 homologs H6 and H9 showed expression only in flowers. Expression profile was also examined in plants growing under phosphate-deficient conditions. Above-ground tissues of the pho1 mutant are constantly under Pi stress due to the deficient transfer of Pi from the roots to the leaves (Poirier et al., 1991
The expression profile of the PHO1 gene family was further analyzed by the promoter-GUS approach. Fragments of 0.5 kb and 1.0 kb of each promoter were cloned in front of the GUS gene and expressed in transgenic plants. The GUS expression profile largely corroborated the RT-PCR results at the whole tissue level. For example, expression of the GUS reporter from the H6 and H9 promoters was restricted to flowers, in agreement with the RT-PCR results. For H6, GUS expression was confined to the connective tissues of the stamen, while for H9 GUS was found solely in the pollen grains (Fig. 9). In some cases, RT-PCR appeared to be more sensitive than the promoter GUS approach. For example, no GUS staining could be obtained with the H2 promoter, while RT-PCR revealed weak expression in a range of tissues (Fig. 8).
Several distinct GUS expression patterns have been found for various organs (Fig. 9; Table II). For roots, the H1, H3, H5, H7, and H8 were expressed in the vascular cylinder, while for H4 and H10 GUS expression was rather localized in the epidermal/cortical cells. Expression in the root tip has also been observed for H5 and H7. In leaves, GUS expression has been found in the vascular cylinder (H1 and H3), the hydathodes (H4, H7, H8, and H10), the trichomes and cells at the base of trichomes (H8), associated with the petiole (H5 and H8), or as a diffuse pattern across the leaf blade (H10). Expression in stems can be observed either associated primarily with the vascular cylinder (H1 and H8) or as a more diffuse pattern (H5, H7, and H10). Several tissues have been found to express GUS in flowers, including the pollen grains (H4, H8, H9, and H10), the vascular cylinder of sepals (H1 and H10), petals (H1) or filament (H1, H3, H5, H7, and H10), the apical end of the stigma excluding the papillae (H1, H3, H4, H5, H7, H8, and H10), the receptacle (H1, H3, H5, H7, H8, and H10), and the connective of the anthers (H1, H5, and H6). In the case of pollen grains, GUS staining could also be observed in the pollen tubes of germinating grains, indicating that GUS expression was not limited to the exine layer. Expression of the H4, H8, H9, and H10 genes in purified pollen grain was also detected by RT-PCR (data not shown).
The Arabidopsis genome database reveals the presence of 10 genes homologous to PHO1, although EST or cDNA are only described for four members of the family, namely PHO1 and the three homologs H1, H2, and H5. Our analysis of the expression pattern of all PHO1 family members by RT-PCR as well as cloning and sequencing of cDNAs revealed that all PHO1 members are expressed. It has been observed for PHO1 as well as for several PHO1 homologs that attempts to clone full-length cDNA directly into E. coli using plasmids, such as pBluescript KS+, consistently resulted in the recovery of aberrant clones representing either incomplete spliced products or having E. coli transposon inserted into the gene, resulting in the truncation of the open-reading frame (Y. Wang, D. Hamburger, and Y. Poirier, unpublished data). It is speculated that expression of mRNA from cryptic promoters on bacterial plasmids resulting in production of intact proteins of PHO1 and its homologs is toxic to E. coli. By contrast, PHO1 can be well expressed in yeast (Hamburger et al., 2002  ). All PHO1 homologs have thus been cloned through direct transformation into yeast, resulting in the recovery of clone encoding full-length protein. The potential toxicity of proteins belonging to the PHO1 family in E. coli is likely to be one cause of the poor representation of full-length cDNAs, despite the apparent broad expression of the corresponding genes in various tissues.
All members of the PHO1 protein family share a number of basic features. All proteins are of similar length, from 745 amino acids for H4 to 823 amino acids for H5, and are formed of two distinct parts, i.e. theN-terminal half being mainly hydrophilic, while theC-terminal half shows the presence of at least six potential membrane-spanning domains (Hamburger et al., 2002 The level of similarity between the members of the PHO1 protein family is relatively high, with theN-terminal hydrophilic half being responsible for most of the divergence. Interestingly, the divergence of the hydrophilic domains correlates with a greater divergence in the exon/intron structure at the 5' end of the genes. Phylogenetic analysis of the PHO1 protein family revealed the presence of three clusters (Fig. 3). In some cases, the phylogenetic relationship can be well correlated with the distribution of the genes on the genome. This is the case for the cluster regrouping the H2, H3, H5, and H6 genes, which is linked to several duplication events implicating chromosomes 1 and 2. It is too early to draw conclusions between the relevance of the phylogenetic relationship and the biological role of the PHO1 homologs belonging to a cluster, since a mutant phenotype has been described so far only for PHO1. The expression pattern of members of a cluster can be significantly divergent, with, for example, H3 being expressed in the vascular tissue of leaves and roots, while H6 is only expressed in connective of the anther (Fig. 9; Table II). It is however striking that the two rice homologs that have been identified have higher similarity to the cluster containing PHO1 and the H1 than the level of similarity between the PHO1/H1 proteins and any other PHO1 homolog within the Arabidopsis genome. This level of conservation across monocots and dicots suggests that the function of the rice homologs and the Arabidopsis PHO1 and H1 are likely to be similar.
All members of the PHO1 family have, within theN-terminal hydrophilic half, the SPX domain divided into three subdomains. While in most eukaryotic proteins the three SPX subdomains are found close together, in the PHO1 protein family these subdomains are separated by somewhat large regions (80–160 amino acids) of low similarity. Interestingly, the region of high similarity in the N-terminal hydrophilic region extends beyond the SPX domain to include the region between SPX-3 and the start of the first putative transmembrane-spanning domain (approximately 50 amino acids; Fig. 2). Although the function of the SPX domain is at present unknown, it is interesting to note that in yeast several proteins implicated in phosphate transport or sensing have a SPX domain, namely the cyclin-dependent kinase inhibitor PHO81, a key regulator of the yeast PHO operon, as well as the PHO87, PHO90, and PHO91 proteins involved in low-affinity Pi transport and potentially Pi sensing (Lenburg and O'Shea, 1996
The SPX domain is also found in yeast in the VTC1, VTC2, VTC3 and VTC4 proteins forming a membrane chaperone complex involved in the sorting of proteins to the vacuolar membrane, including the V-ATPase (Cohen et al., 1999 In addition to all members of the PHO1 family, nine other proteins have been identified in Arabidopsis that have a SPX domain. These proteins can be grouped into three families distinct from the PHO1 family (Fig. 6d), with only one of them having features of integral membrane proteins. Unfortunately, the function of these nine proteins are presently unknown but could eventually provide some clues on the role of the SPX domain in plants.
Among proteins in eukaryotes having a SPX domain, only few of them also possess an EXS domain. In yeast, only SYG1 has both SPX and EXS, while in Arabidopsis only the members of the PHO1 family possess both domains. The EXS domain in the PHO1 proteins encompasses approximately half of the hydrophobic region. The function of the EXS domain is also unknown, although it is interesting to note that the yeast ERD1 protein, which possesses a EXS domain, is involved in the localization of endogenous endoplasmic reticulum proteins, further reinforcing the connection between SPX and EXS with sorting of protein to endomembranes (Hardwick et al., 1990
Analysis of the expression pattern of all PHO1 homologs reveals that a broad range of tissues can express members of the PHO1 family. However, expression of PHO1 genes in the vascular tissue appears to be predominant. For example, out of the eight genes that are expressed in roots as revealed by both GUS expression and RT-PCR, six demonstrated a GUS expression pattern in the root vascular tissue similar to the pattern initially reported for PHO1 (Fig. 9; Hamburger et al., 2002
The function of the PHO1 gene family is however clearly not limited to Pi movement to or from the vascular tissues. This is perhaps most clearly revealed by the expression of several homologs in the pollen grain (H4, H8, H9, and H10) but also indicated through the expression of the GUS protein in tissues not closely connected to the vascular tissue, such as expression of the H4 and H10 promoters in the epidermal cortical cells of the roots. In pollen, expression of PHO1 homologs could be related to the import of Pi into the maturing pollen since plasmodesmatal connections between developing pollen and the tapetum are lost early in development (Owen and Makaroff, 1995
Analysis of the pho1 mutant combined with the expression pattern of the PHO1 gene revealed a role of PHO1 in the loading of Pi to the xylem vessels, implicating that PHO1 could be mediating the exit of Pi out of the xylem parenchyma cells (Poirier et al., 1991
Plant Material
Leaves, stems, flowers, or pollen were harvested from wild-type Arabidopsis, accession Columbia, or the pho1-3 mutant, grown in pots in fertilized soil under continuous illumination at 19°C (Poirier et al., 1991
Total RNA from leaves, flowers, pollen, or roots was isolated by the Trizol method (Chomczynski and Sacchi, 1987
Analysis of the expression profile was done by semiquantitative RT-PCR based on the protocol of Burleigh (2001)
Genomic fragments of 0.5 and 1.0 kb located upstream of the PHO1 transcribed sequence were isolated by PCR using oligonucleotides, creating a novel HindIII site at the 5' end and either a XbaI or BamHI site at the 3' end. Each fragment was cloned in front of the GUS gene in the binary vector pBI121 (Malik and Wahab, 1993 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY507953 and AY507962.
We thank Aleksandra Stefanovic and Lassaad Belbahri for critical reading of the manuscript and Simon Goepfert for help with the ClustalX alignment. Received December 17, 2003; returned for revision February 2, 2004; accepted February 4, 2004.
1 This work was supported by the Fonds National Suisse de la Recherche Scientifique (grant no. 31–61731.00 to Y.P.). Contributions are also acknowledged from the University of Lausanne and the Canton de Vaud. 
2 Present address: Nutrition and Health Department, Functional Microbiology Group, Nestlé Research Center, P.O. Box 44, CH–1000 Lausanne 26, Switzerland.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.037945. * Corresponding author; e-mail yves.poirier{at}ie-bpv.unil.ch; fax 41–21–692–4195.
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subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature 325: 321–326
subunit deficiency by interacting with the 
