Identification of two conserved cis-acting elements, MYCS and P1BS, involved in the regulation of mycorrhiza-activated phosphate transporters in eudicot species


Author for correspondence:
Guohua Xu
Tel: +86 25 84396246


  • In this study, six putative promoter regions of phosphate transporter Pht1;3, Pht1;4 and Pht1;5 genes were isolated from eggplant and tobacco using the inverse polymerase chain reaction (iPCR). The isolated sequences show evolutionary conservation and divergence within/between the two groups of Pht1;3 and Pht1;4/Pht1;5.
  • Histochemical analyses showed that all six promoter fragments were sufficient to drive β-glucuronidase (GUS) expression specifically in arbuscular mycorrhizal (AM) tobacco roots and were confined to distinct cells containing AM fungal structures (arbuscules or intracellular hyphae).
  • A series of promoter truncation and mutation analyses combined with phylogenetic footprinting of these promoters revealed that at least two cis-regulatory elements – the mycorrhiza transcription factor binding sequence (MYCS) first identified in this study and P1BS – mediated the transcriptional activation of the AM-mediated inorganic phosphate (Pi) transporter genes. Deletion or partial mutation of either of the two motifs in the promoters could cause a remarkable decrease, or even complete absence, of the promoter activity.
  • Our results propose that uptake of inorganic phosphate (Pi) by AM fungi is regulated, at least partially, in an MYCS- and P1BS-dependent manner in eudicot species. Our finding offers new insights into the molecular mechanisms underlying the coordination between the AM and the Pi signalling pathways.


Phosphorus (P) is an essential macronutrient for plant growth and development. The primary source of P taken up by plants is inorganic phosphate (Pi) in soils (Raghothama, 1999). As a result of chemical fixation and the formation of organic complexes, P is one of the least available plant nutrients in soil, and is often a limiting factor to crop yields (Schachtman et al., 1998; Abel et al., 2002). One of the evolutionary adaptations of plants to low Pi supply is the formation of symbiotic associations with arbuscular mycorrhizal (AM) fungi (Harrison, 2005). Arbuscular mycorrhizal symbiosis is an ancient reciprocal association that originated several hundred million years ago, and > 80% of terrestrial plants in natural ecosystems are able to develop AM roots (Remy et al., 1994; Schüssler et al., 2001; Smith & Read, 2008).

Plant cells need to maintain cytoplasmic Pi concentrations at a millimolar range, but the Pi concentration in soil solution rarely exceeds 10 μM (Schachtman et al., 1998). Specific transport systems are essential for taking up Pi and distributing it within plants (Raghothama, 1999; Smith & Barker, 2002; Javot et al., 2007b). In nonmycorrhizal plants, Pi from the rhizosphere is taken up directly by plant roots mainly via high-affinity Pi transporters (PiTs) that are localized in the epidermis and root hairs. After colonization, AM plants have two Pi-uptake pathways, either direct uptake, via the roots at the plant–soil interface, or indirectly, via the AM or symbiotic uptake, at the plant–fungal interface. The latter is often the dominant pathway (Smith et al., 2003, 2004).

The identification and subsequent characterization of AM-responsive genes is the first step towards understanding the molecular mechanism of transcriptional induction upon mycorrhiza development (Salzer et al., 1999; Frenzel et al., 2006). In recent studies, several AM-inducible PiT genes belonging to the Pht1 family in AM roots have been isolated from diverse plant species (Javot et al., 2007b). These are StPT3, StPT4 and StPT5 in potato (Solanum tuberosum) and LePT3, LePT4 and LePT5 in tomato (Solanum lycopersicum) (Rausch et al., 2001; Nagy et al., 2005; Xu et al., 2007) and their orthologous genes in pepper, tobacco, eggplant and petunia (Chen et al., 2007a; Wegmüller et al., 2008), OsPT11 and OsPT13 in rice (Oryza sativa) (Paszkowski et al., 2002; Glassop et al., 2007), MtPT4 in Medicago truncatula (Harrison et al., 2002; Javot et al., 2007a), LjPT3 and LjPT4 in Lotus japonicus (Maeda et al., 2006; Guether et al., 2009), HORvu;Pht1;8 in barley (Hordeum vulgare), TRIae;Pht1;Myc in wheat (Triticum aestivum) and ZmPT6 in maize (Zea mays) (Glassop et al., 2005). It is interesting to note that the promoter regions of StPT3 and MtPT4 were capable of directing the expression of the β-glucuronidase (GUS) reporter gene in AM roots of several eudicot species (Karandashov et al., 2004). Knockdown of LjPT3 and knockout of MtPT4 resulted in similar phenotypes that repressed arbuscule formation during AM symbiosis (Maeda et al., 2006; Javot et al., 2007a). These findings suggest a conserved mycorrhiza-regulated pathway in different AM-forming species (Karandashov & Bucher, 2005; Javot et al., 2007b).

Previous studies have demonstrated that the promoters of StPT3, StPT4 and LePT4 genes are able to drive AM-regulated expression of the GUS reporter gene in cortical colonized cells (Karandashov et al., 2004; Nagy et al., 2005). The identification of promoter elements or genes encoding transcription factors mediating the transcriptional induction of AM-responsive PiTs remains a major challenge. Although several transcription factors, such as AtPHR1 (OsPHR2), OsPTF1, AtBHLH32, AtWRKY75, AtZAT6 and AtMYB62, have been reported to be involved in the adaptation of plants to low-P conditions and shown to regulate the expression of a subset of Pi starvation-inducible genes (Rubio et al., 2001; Yi et al., 2005; Chen et al., 2007b; Devaiah et al., 2007a,b, 2009; Zhou et al., 2008), only the PHR1-binding cis-acting element P1BS (GNATATNC) has been identified to be involved in the activation of Pi starvation-inducible genes, including PiTs (Rubio et al., 2001; Schünmann et al., 2004). Little is known about the mechanisms leading to the transcriptional activation of PiT genes in response to AM symbiosis.

The aim of this study was to identify cis-elements involved in the activation of AM-regulated PiT expression, thus facilitating the isolation and functional characterization of the transcription factor(s) responsible for AM-regulated plant genes in future work. In our previous work, we cloned PiT genes belonging to the Pht1 family from several solanaceous species and characterized the conservation and divergence of their coding sequences, as well as their expression patterns, in response to Pi starvation and inoculation by AM fungi (Chen et al., 2007a; Xu et al., 2007). In the present study, we report six AM-regulated PiT promoters of Pht1;3, Pht1;4 and Pht1;5 from tobacco and eggplant. Functional analysis of these promoters by successive truncation and targeted mutation or deletion led to the identification of two conserved elements, P1BS and mycorrhiza transcription factor binding sequence (MYCS), both of which are required to confer the high-level AM-inducible transcription of PiT genes.

Materials and Methods

Plants, cultivation conditions and inoculations

Tobacco (Nicotiana tabacum L. cv Yunyan) and eggplant (Solanum melongena L. cv Suqi) were used in this study. Seeds were surface-sterilized, germinated and maintained in tissue culture using MS medium (Murashige & Skoog, 1962) supplemented with 2% sucrose. The aseptic plants were either transformed with Agrobacterium tumefaciens or transferred to pot culture for inoculation with AM fungi.

In pot culture, the wild-type and transgenic plants were maintained in sand, as described previously (Chen et al., 2007a). Briefly, two plantlets were transplanted to a 3-dm3 plastic pot filled with sterilized sand. A liquid inoculum containing Glomus intraradices (BEG141; Premier Tech Biotechnologies, Quebec, Canada) was used for colonization. Each plantlet was inoculated with 1 ml of inoculum or autoclaved inoculum (as a control), which was placed in the sterilized sand around the roots. The irrigating nutrient solution contained the following macro- and micronutrients: 2 mM KNO3, 1 mM NH4NO3, 0.5 mM Ca(NO3)2, 0.25 mM CaCl2, 0.5 mM MgSO4, 20 μM Fe-EDTA, 9 μM MnCl2, 46 μM H3BO3, 8 μM ZnSO4, 3 μM CuSO4, 0.03 μM (NH4)2MoO4 and either 0.05 mM NaH2PO4 (low-P treatment) or 0.5 mM NaH2PO4 (high-P treatment). The experiment comprised 8–17 replicates for each treatment. The plants were grown in a growth chamber with a 14-h light period at 28–30°C and a 10-h dark period at 18–20°C. Plants were harvested 6 wk after inoculation. The freshly collected roots were washed with deionized water and part of the roots were immediately frozen in liquid nitrogen and stored at −80°C for subsequent RNA isolation. Other parts of the roots were used to evaluate colonization, to assess GUS activity (by histochemical staining) and for visualization of intraradical fungal mycelium.

Isolation of Pht1;3, Pht1;4 and Pht1;5 promoters from tobacco and eggplant

The promoter regions of tobacco and eggplant (Pht1;3, Pht1;4 and Pht1;5) were isolated using the Inverse PCR technique (Nagy et al., 2005). High-molecular-weight genomic DNA was divided into aliquots (five micrograms for each aliquot), each aliquot was digested with EcoRV, DraI, PvuII or StuI, respectively, self-ligated and at least two sets of primers (Supporting Information Table S1) were used for nested PCR of each promoter. The amplified fragments were purified and cloned into the pMD18-T vector (TaKaRa Biotechnology, Dalian, China) for sequencing (Invitrogen Biotechnology). Identification of these promoters was established by the presence of an overlap sequence with the known coding region of the genes (Chen et al., 2007a).

Total RNA extraction and reverse transcription–polymerase chain reaction

Total RNA was extracted from 100 mg of tissue samples using the guanidine thiocyanate extraction method with Trizol reagent (Invitrogen, Reverse transcription–polymerase chain reaction (RT-PCR) was performed, as described earlier (Chen et al., 2007a), using gene-specific primers (Table S2). The GenBank accession numbers for SmPT3, SmPT4, SmPT5, NtPT3, NtPT4 and NtPT5 are EF091668, EF091671, EF091674, EF091669, EF091672 and EF091675, respectively.

Construction of the Pht1;3, Pht1;4 and Pht1;5 promoter deletion series

The isolated fragments of Pht1;3, Pht1;4 and Pht1;5 promoters upstream of the translation initiator, ATG, were amplified via PCR using the primers (Table S3) to the introduced HindIII and BamHI restriction sites (underlined by single and double lines, respectively) at the end of the 5′ and 3′ regions. After digestion with the two restriction enzymes, the amplified fragments were purified and cloned into the linearized binary vector, pBI121, to replace the CaMV35S promoter in front of the GUS gene. The resulting gene constructs were designated pNtPT3, pNtPT4, pNtPT5, pSmPT3, pSmPT4 and pSmPT5. Besides the overall promoter sequence isolated, a series of 5′ truncations of these six promoter fragments was also created using the same strategy. The resulting gene constructs were designated pNtPT3/4/5n or pSmPT3/4/5n. The targeted deletion of the predicted MYCS sequence and mutation of P1BS were generated by overlap and extension PCR (the primers used are listed in Table S4) for the promoters of SmPT4/NtPT5 and NtPT4, respectively. The mutated promoter sequences were cloned into the HindIII- and BamHI-digested pBI121 binary vector to drive GUS gene expression. All of these new derived binary vectors were introduced into A. tumefaciens strain EHA105 for transformation.

Histochemical GUS assays and detection of mycorrhizal fungal colonization

Histochemical staining of the fresh transgenic tobacco root materials for GUS activity was performed as described previously (Karandashov et al., 2004). For visualization of fungal structures, the Magenta-GUS-stained root sectors were treated for 1 h with 1.8 M KOH solution heated to 90°C to clear the tissues, and then treated with 1% HCl (v/v) solution for 5 min. The roots were counterstained for 2 h at 90°C with 0.3% Trypan Blue (dissolved in lactoglycerol (lactic acid/glycerol/water, 1 : 1 : 1, v/v/v)) (Philips & Hayman, 1970; David-Schwartz et al., 2001). The co-localization of Magenta-GUS and Trypan Blue stains were indicated by purple staining of the sections The stained material was rinsed in 50% glycerol to remove excess stain before being photographed under a stereomicroscope using a colour charge-coupled device (CCD) camera (Olympus Optical Co. Ltd, Tokyo, Japan). A visual-based quantification method (the percentage of roots showing induced GUS activity) (Kosuta et al., 2003; Gleason et al., 2006; Drissner et al., 2007) was used to evaluate GUS activity for different promoter truncation/deletion constructs. Briefly, > 100 root segments (1–2 cm), cut from individual transgenic plants, were stained for 6 h, and the number of root segments showing GUS induction and the total number of root segments were counted. For each construct, three independent experiments, each with 8–17 individual plants, were analyzed using these histochemical methods.

For enzymatic GUS assays, root tissues were ground in liquid nitrogen and transferred to microtubes containing 1 ml of the extraction buffer (50 mM sodium phosphate, pH 7.5, 10 mM Na2EDTA, 10 mM 2-mercaptoethanol, 0.1% Triton X-100 and 0.1% (w/v) sodium lauryl-sarcosine) for total protein extraction. Beta-glucuronidase activity was measured fluorimetrically using 1 μg of total protein extract, as described previously (Andriankaja et al., 2007).

Bioinformatics analysis

Multiple sequence alignment was performed using the GeneTool Multi-Align Editor program (BioTools, Inc., Edmonton, AB, Canada). Phylogenetic tree analysis was performed using the MEGA phylogeny program ( Analyses of known cis-regulatory motifs in the promoters were performed using the PLACE database ( (Higo et al., 1999). Predicted conserved motifs were screened within the promoter regions of many orthologous PiTs from diverse species using the FootPrinter 3.0 algorithm web server, as described previously ( (Gumucio et al., 1992; Karandashov et al., 2004). The motifs were identified using a motif size of 8–12, allowing for one pair mutation or motif losses in a subregion of 100. The identified motifs were then scanned in the corresponding promoters using the RSA-tools DNA-pattern matching program (


Identification and analysis of the AM-responsive PiT promoters

We have previously identified and characterized five Pht1 genes from three solanaceous species: pepper, eggplant and tobacco (Chen et al., 2007a). Expression and phylogenetic analysis demonstrated that three of the five Pht1 genes in each species, Pht1;3-5, were upregulated by mycorrhiza and can be clustered into two groups, consisting of Pht1;3 and Pht1;4/Pht1;5 (Fig. S1).

To gain further insights into the regulatory mechanism underlying the AM-enhanced expression, the putative promoter fragments immediately upstream of the translation start ATG were isolated for Pht1;3, Pht1;4 and Pht1;5 from tobacco and eggplant using inverse polymerase chain reaction (iPCR): 430 bp for SmPT3, 532 bp for NtPT3, 865 bp for SmPT4, 962 bp for NtPT4, 2098 bp for SmPT5 and 444 bp for NtPT5 (Fig. S2). Based on the Pht1 complementary DNA (cDNA) sequences obtained using Rapid Amplification of cDNA Ends – Polymerase Chain Reaction (RACE – PCR), the transcriptional start site (TSS) could be easily distinguished and was found to be very close to the ATG start codon in all six promoters (Fig. S2). The conserved core promoter element of eukaryotes, the TATA-box, was identified −25 to –30 bp upstream of the TSS in all six promoter sequences, whereas another promoter consensus CAAT-box was found between −7 and 103 bp from its TSS (Fig. S2).

Promoter cis-element analysis using the PLACE algorithm ( revealed that a known regulatory motif, P1BS, an element associated with Pi starvation signalling, a root motif box (ATATT or AATAT) and an unknown element (TCTTGTT) are present in all six promoter regions (Fig. S2). Some of the promoters contain two other motifs – NODCON2GM (CTCTT) and OSE1ROOTNODULE (AAAGAT) – which are conserved elements associated with AM- and nodule-induced leghaemoglobin gene regulation (Stougaard et al., 1990; Vieweg et al., 2004; Fehlberg et al., 2005). Unlike the StPT3 promoter, the six promoter regions do not contain miniature inverted repeat transposable element (MITE) and AM- and resistance-related (MRR) elements (Rausch et al., 2001) (Fig. S2).

Nucleotide sequence alignment between the nine promoters of AM-regulated Pht1 genes in solanaceous species showed the presence of several conserved regions (Fig. 1). The promoters of Pht1;3 members (SmPT3, NtPT3 and StPT3) and Pht1;4 members (SmPT4, NtPT4, LePT4 and StPT4) were found to share a consecutive 152–155 bp fragment with 76.7–83.7% identity (green in Fig. 1) and a 203–206 bp fragment with 79.8–94.1% identity (red and dark blue in Fig. 1). In addition, the promoters of NtPT5 and SmPT5 also shared a 166–177 bp fragment with 79.5% identity (red in Fig. 1). Interestingly, the longer conserved region of Pht1;3 members in the StPT3 promoter was located at −618/−460 (green in Fig. 1), including part of the 129 bp fragment (−639/−510) of the StPT3 promoter, which has previously been revealed to be responsible for the AM-mediated up-regulation (Karandashov et al., 2004). However, in the SmPT3 and NtPT3 promoters, the conserved region was located within a 300 bp region upstream of the translational start codon, suggesting a probable promoter rearrangement or insertion during the evolution of StPT3. In addition, poor identities were observed in regions other than the conserved region between all the Pht1;3, Pht1;4 and Pht1;5 promoters. The conserved expression pattern (AM enhancement) of Pht1;3-5 leads to the hypothesis that these conserved regions may contain essential elements responsible for AM-activated expression. In addition, the isolated promoter sequences were overall highly divergent between the two groups of Pht1;3 and Pht1;4/Pht1;5, whereas those within the group of Pht1;4/Pht1;5 were similar to some extent (red in Fig. 1).

Figure 1.

 Schematic sequence comparison of the promoters for the inorganic phosphate transporter (PiT) genes Pht1;3, Pht1;4 and Pht1;5 in solanaceous species. The transporters and corresponding plant species are: StPT3, StPT4 and StPT5 from potato (Rausch et al., 2001; Karandashov et al., 2004; Nagy et al., 2005); LePT4 from tomato (Nagy et al., 2005; Xu et al., 2007); SmPT3, SmPT4 and SmPT5 from eggplant and NtPT3, NtPT4 and NtPT5 from tobacco (Chen et al., 2007a). Corresponding promoter regions shaded in the same colour below the coordinate axis represent structural commonalities (> 70% sequence identity) between these promoters. Promoter regions shaded in different colours or with intervening sequences do not display obvious similarity. The six promoters isolated in this study are shown in italics.

The isolated fragments of six PiT promoters are sufficient to drive AM-dependent expression of GUS in transgenic tobacco

As the Pht1;3, Pht1;4 and Pht1;5 promoter sequences isolated from tobacco and eggplant differed widely in length, we first examined if these promoter fragments were sufficient to confer their AM-responsive expression. Each of the six promoters was amplified and fused to the GUS reporter gene, and introduced into tobacco plants via A. tumefaciens-mediated transformation. The individual transgenic plants were subsequently inoculated or not with Glomus intraradices. Six weeks after colonization, transcripts of NtPT3, NtPT4 and NtPT5, the three AM symbiosis-responsive genes (Chen et al., 2007a), were found to be abundant (as detected by RT-PCR) in the tobacco roots (Fig. 2a).

Figure 2.

 Functional analysis of Pht1;3, Pht1;4 and Pht1;5 promoters isolated from eggplant and tobacco. (a) Molecular detection for mycorrhizal symbiosis of the transgenic tobacco roots by reverse transcription–polymerase chain reaction (RT-PCR) using NtPT3, NtPT4 and NtPT5 as markers. (b–g) Localization of β-glucuronidase (GUS) activity in mycorrhizal tobacco root sectors driven by the promoters of SmPT3, SmPT4 and SmPT5 from eggplant (b–d, respectively) and by the promoters of NtPT3, NtPT4 and NtPT5 from tobacco (e–g, respectively). (h–k) Co-localization of GUS activity (indicated by the purple colour, from the overlay of the Trypan Blue and Magenta-GUS stains) shows that SmPht1;3 (h), SmPht1;4 (i, j) and SmPht1;5 (k, l) are exclusively active in hyphae-colonized cortical cells. Individual arbuscule structure was dissected from mycorrhizal roots carrying the pSmPT5::GUS chimeric gene (k). Red arrows indicate arbuscule or arbusculate hyphae, white arrows indicate intracellular hyphae, green arrows indicate vesicles and yellow arrows indicate noncolonized cells. Bars, 50 μm.

As observed, GUS activity could be detected only in distinct areas of AM transgenic roots for all the six chimeric genes (pSmPT3-430::GUS, pNtPT3-532::GUS, pSmPT4-865::GUS, pNtPT4-962::GUS, pSmPT5-2098::GUS and pNtPT5-444::GUS) (Fig. 2b–g,h–l). Co-localization of GUS expression and AM fungal structure by overlay of Magenta-GUS with Trypan Blue staining demonstrated that GUS activity driven by Pht1;3, Pht1;4 and Pht1;5 promoters from eggplant was confined to distinct cortical cells corresponding to the fungal colonization (Fig. 2h–l). No GUS staining was observed in vesicle-containing cells or in noncolonized cells. These results revealed that all six promoter fragments contain essential cis-regulatory elements which are necessary to confer AM- responsive expression.

MYCS is a functional cis-element mediating AM-regulated PiT gene expression

In order to identify the cis-elements located within the functional regions of the promoters that are required for AM-regulated gene expression, phylogenetic footprinting combined with sequence comparative analysis was performed to screen candidate regulatory elements associated with AM-activated expression in different PiT promoter sequences. As shown in Figs S2, 3(a,b), a consensus sequence (TTTCTTGTTCT with a maximum variation of one nucleotide) was identified exclusively in all promoter regions of 10 AM-regulated PiTs from eudicot solanaceous species and from Medicago truncatela (MtPT4). There is only one copy of the element present in all these regions with one nucleotide variation (Fig. S2). We designated this novel conserved element in the promoters of AM-activated PiT genes in eudicot species as MYCS. Apart from its location, −535 bp upstream from the translation start codon, ATG, in the StPT3 promoter, in all other promoters MYCS is located close to the P1BS motif and to the ATG start codon (−95 to −174 bp upstream). Sequence comparative analysis revealed that MYCS is highly similar to a putative element named CTTC-motif (cttcttgttcta), which was identified within the 129 bp regulatory region of the StPT3 promoter (Karandashov et al., 2004). No such motif is present in the promoters of AM-specific OsPT11 from monocot rice, and Pi-starvation up-regulated PiT genes including AtPT8, OsPT2 and SmPT2, which were also isolated in this study (Fig. 3a).

Figure 3.

 Putative cis-regulatory elements in the promoters of plant inorganic phosphate transporter (PiT) genes. (a) The putative cis-regulatory elements were screened using phylogenetic footprinting combined with DNA-pattern matching analysis within the region of six newly isolated promoters (shown in italics) and the promoter regions of other mycorrhiza-inducible PiTs from tomato (LePT4) and potato (StPT3 and StPT5), Medicago truncatula (MtPT4) and monocot Oryza sativa (OsPT11). The promoters of Pi starvation-inducible PiTs, AtPT8 and SmPT2 from dicot Arabidopsis thaliana and eggplant, and OsPT2 from O. sativa, were used as controls. The fragment of the SmPT2 promoter was also isolated in this study. Sequences are ordered automatically by the program FootPrinter 3.0 ( according to the phylogenetic relationship of the corresponding coding regions, as shown on the right. PIBS, GNATATNC; mycorrhiza transcription factor binding sequence (MYCS), TTTCTTGTTCT, or with one nucleotide variation; PHO, CACGTG; W-box, TTGACY. (b) One copy of the MYCS motif is presented in the promoter regions (95 to 535 bp upstream from the translation start codon, ATG) of all 10 mycorrhiza-regulated PiT genes in eudicot species. The red letters represent the one-nucleotide-variation in each MYCS, if any.

In an attempt to determine whether MYCS is essential for the AM-activated expression of Pht1;3, Pht1;4 and Pht1;5 genes in solanaceous species, we performed functional analyses of successive truncations of the six promoters. We designed a series of constructs containing the MYCS motif and introduced them into transgenic tobacco plants. For each construct, more than eight independent transgenic lines were inoculated with G. intraradices and analyzed. As observed, the truncated fragments (pSmPT3-154, pSmPT4-174, pSmPT5-202, pNtPT3-225, pNtPT4-267 and pNtPT5-199) (Fig. 4a), which were cut within the conserved regions containing the MYCS in each promoter (Fig. 1), were sufficient to direct GUS reporter gene expression exclusively in AM transgenic roots (Fig. 4a). The overall expression pattern in response to AM fungal colonization driven by the truncated short promoters was similar to the activation of the GUS reporter by the whole-length fragment of these six promoters (Fig. 2b–g).

Figure 4.

 Functional analyses of the conserved mycorrhiza transcription factor binding sequence (MYCS) motif involved in mycorrhiza-regulated activation. (a) Sequence comparison of the narrowed regulatory regions in the promoters of Pht1;3, Pht1;4 and Pht1;5 genes from eggplant and tobacco, and β-glucuronidase (GUS) staining in mycorrhizal tobacco roots driven by the chimeric promoters with different 5′ deletions. For clear observation of the root tissues, the root segments were treated for 1 h with 1.8 M KOH solution at 90°C after GUS staining. MYCS (TTTCTTGTTGT, or with one nucleotide variation) and P1BS (GNATATNC) motifs are shown in red and blue, respectively. (b) Schematic illustration of the constructs of pSmPT4-132, pSmPT4-174-M and pNtPT5-199-M and GUS staining in mycorrhizal tobacco roots driven by the three constructs. Approximate promoter lengths are given with distance from the ATG start codon. MYCS and P1BS motifs are highlighted in red and blue, respectively. −M indicates the MYCS deletion. The relative strength of GUS activity was screened visually in independent tobacco roots (see the Materials and Methods section) and is presented as the percentage of mycorrhizal roots exhibiting induced GUS activity. The GUS activity in protein extracts from individual roots was measured quantitatively using fluorimetric assays. For each construct, three separate experiments, each containing 8–17 individual plants, were analyzed. Error bars indicate SD.

As the MYCS is present in the conserved 3′ proximal region of the promoters (Fig. 3a), we generated two other constructs with deletion of this motif (TTTCTTGT) from pSmPT4-174 and pNtPT5-199, respectively, by overlap and extension PCR (Fig. 4b). Histochemical staining for GUS revealed that deletion of MYCS (pSmPT4-174-M and pNtPT5-199-M) resulted in the almost complete absence of GUS activity in the transgenic lines (Figs 4b, S3b,c), even though the total AM colonization rate was not significantly different (Fig. S3d). This finding was also supported by the observation that the pSmPT3-132 chimeric gene not containing the MYCS motif strongly suppressed GUS activity in its AM transgenic roots (Figs 4b, S3a). These data highlight the functional role of MYCS for the AM-inducible response of Pht1 genes in solanaceous species.

The conserved P1BS element is also involved in the regulation of AM-activated PiT gene expression

To further investigate the role of the MYCS in directing the AM-regulated expression of PiTs, additional 5′-end deletions within the proximal regulatory regions of the six promoters were generated. Interestingly, as in pSmPT4-174-M and pNtPT5-199-M, deletions of pSmPT4 down to −148 (pSmPT4-148) or of pNtPT5 down to −175 (pNtPT5-175), resulted in a strong decrease, or even complete abolition, of GUS activity (Figs 5a, S3e,f), although both promoter truncations still contained the MYCS (Fig. 5a). This finding suggests that the 26-bp region (−174/−148) of the SmPT4 promoter and the 24-bp region (−199/−175) of the NtPT5 promoter contain other motif(s) necessary for the AM-inducible activation.

Figure 5.

 Functional analysis of the conserved P1BS motif involved in mycorrhiza-regulated activation. (a) Schematic illustration of the pSmPT4-148 and pNtPT5-175 constructs and the β-glucuronidase (GUS) activity in mycorrhizal tobacco roots driven by the two constructs. Mycorrhiza transcription factor binding sequence (MYCS) and P1BS motifs are shown in red and blue, respectively. The relative strength of GUS activity was screened visually in individual tobacco roots and is presented as the percentage of mycorrhizal roots exhibiting induced GUS activity. The GUS activity in protein extracts from individual roots was measured quantitatively using fluorimetric assays. For each construct, three separate experiments, each containing 8–17 individual plants, were analyzed. Error bars indicate standard deviations. (b) Co-localization of Magenta-GUS staining and Trypan Blue staining of mycorrhizal structures in the mycorrhizal transgenic tobacco roots harboring the pNtPT4-267–GUS chimeric gene under both low-inorganic phosphate (Pi) (−P + M, 0.05 mM Pi) and high-Pi (+P + M, 0.5 mM Pi) conditions. Red arrows indicate arbuscule or arbusculate hyphae; the green arrow indicates intracellular hyphae. (c) Schematic illustration of the constructs of pNtPT4-267 and pNtPT4-267-P1BS-mu and the GUS activity in mycorrhizal tobacco roots driven by the two constructs. MYCS and P1BS motifs are highlighted in red and blue, respectively. The mutated P1BS (T to G and A to C transitions, except positions 2 and 7 of P1BS) are shown in green. For clear observation of the root tissues, the GUS-stained root segments were treated for 1 h with 1.8 M KOH solution heated to 90°C. The relative strength of GUS activity was screened visually in individual tobacco roots and is presented as the percentage of mycorrhizal roots exhibiting induced GUS activity. The GUS activity in protein extracts from individual roots was measured quantitatively using fluorimetric assays. For each construct, three separate experiments, each containing 8–17 individual plants, were analyzed. Error bars indicate SD.

The corresponding regions of the other three promoters (−225 to −191 for pNtPT3, −267 to −123 for pNtPT4 and −202 to −131 for pSmPT5; Fig. 4a) were thus subjected to further analysis. Even though MYCS was present in all of these truncated short promoters, histochemical staining revealed that removal of the 34 (−225 to −191), 143 (−267 to −123) and 71 (−202 to −131) bp domains for pNtPT3, pNtPT4 and pSmPT5 respectively, led to almost complete lack of GUS activity in the transgenic tobacco roots. Sequence comparison between these narrowed regulatory regions allowed us to identify a conserved motif, P1BS (GNATATNC), as the only consensus sequence shared by all the six regions (Fig. 4a).

The P1BS motif has been well documented to be a functional phosphate-responsive element (Rubio et al., 2001; Schünmann et al., 2004; Schachtman & Shin, 2007), and we also observed that a high Pi content repressed the GUS activity in transgenic roots harboring the pNtPT4-267–GUS chimeric gene, although a less intensive, but distinct, colonization could be observed (Fig. 5b). This prompted us to explore the possible role of P1BS in the transcriptional activation of AM-responsive PiT genes. As in the case of MYCS, we performed targeted mutation of P1BS (T to G and A to C transitions, except positions 2 and 7 of P1BS) in pNtPT4-267, resulting in pNtPT4-267-P1BS-mu (Fig. 5c). As expected, pNtPT4-267-P1BS-mu could drive only a very low level of expression of GUS in transgenic AM tobacco roots (total 17 individual lines, Fig. 5c). This confirmed the positive role of P1BS in mediating the AM-responsive expression of PiT genes. More interestingly, we found that P1BS is located near the MYCS element in the promoter regions of all mycorrhiza-regulated PiT genes, with only 12–94 bp between the two motifs (Figs 3, 4a). It leads to the suggestion that P1BS may act in combination with MYCS to regulate the mycorrhiza-mediated PiT genes.


In the natural soil ecosystem, most terrestrial plants have evolved an AM Pi-uptake pathway. Identifying the cis-elements in the promoter region of the AM-up-regulated Pht1 genes is an important step towards identifying AM signalling components with significant regulatory roles. In the present work, we reported six AM-mediated PiT promoters, within which the two conserved motifs, MYCS and P1BS, were identified as being involved in the activation of AM-responsive expression.

Conservation and divergence of the AM-regulated PiTs and their promoters in solanaceous species

In recent studies, AM-regulated PiTs, which may be responsible for Pi uptake at the periarbuscule membrane, have been identified in several plant species, mainly in three families: Gramineae, Leguminosae and Solanaceae (Rausch et al., 2001; Harrison et al., 2002; Paszkowski et al., 2002; Glassop et al., 2005, 2007; Nagy et al., 2005; Maeda et al., 2006; Chen et al., 2007a; Xu et al., 2007; Wegmüller et al., 2008). Earlier results, based on the phylogenetic analysis of putative encoded proteins, clustered five Pht1 genes of solanaceous species into three clades, and grouped the three AM-regulated PiT genes into two subclades, namely AM-enhanced Pht1;3 and AM-induced Pht1;4/Pht1;5 (Nagy et al., 2005; Chen et al., 2007a).

The similar expression profiles in response to AM symbiosis, but the relatively remote phylogenetic distance between Pht1;3 and Pht1;4/Pht1;5 in solanaceous species (Fig. S1) prompted us to sequence the promoters of these genes and to investigate their evolutionary relationships. Sequence alignment of the isolated promoters revealed high identities within the subclades, but much fewer structural commonalities between the subclades of Pht1 genes in solanaceous species (Fig. 1). Four members of the Pht1;4 group (StPT4, LePT4, SmPT4 and NtPT4) showed a common, conserved region of c. 200 bp proximal to the start codon ATG (Fig. 3), of which StPT4 and LePT4 promoters have much higher identity in the whole sequence (Nagy et al., 2005). The results support the conclusion that the members of the Pht1 family had diverged before solanaceous species evolved into different plant lineages, and then underwent independent evolution within each lineage (Nagy et al., 2005).

As the isolated Pht1;5 promoters have high structural commonalities with known Pht1;4 promoters (Fig. 2), it might be not surprising that the Pht1;5 members have a tissue-expression pattern similar to that of Pht1;4 members in solanaceous species. We observed that both Pht1;4 and Pht1;5 promoters from eggplant and tobacco drove the GUS reporter exclusively restricted in the colonized cortical cells containing AM fungal structures (arbuscules or intracellular hyphae) (Fig. 2i–l), similar to the AM-inducible expression of LePT4 and StPT4 confined in distinct cells containing the fungal structures in potato (Nagy et al., 2005). However, laser micro-dissection (LMD) analysis showed that LePT5 messenger RNA (mRNA) in tomato was not only present in colonized cells, but also found in noncolonized cells from AM roots (Balestrini et al., 2007). Such discrepancy might be caused by the lower sensitivity of GUS reporter staining in noncolonized root cortex, to the presence of other regulatory elements located beyond the promoter regions that we isolated, or to the functional divergence of Pht1;5 promoters in solanaceous species.

MYCS is a conserved and functional component required for the transcriptional activation of mycorrhiza-inducible Pi transporter genes in eudicot species

It is generally accepted that certain cis-acting elements or degenerated sequence motifs arranged in promoters or introns play pivotal roles in gene regulation and are responsible for the tissue-specific and/or development-dependent expression pattern (Berman et al., 2002; Le Hir et al., 2003; Schallau et al., 2008). As none of the Pht1;3, Pht1;4 and Pht1;5 genes cloned from tobacco or eggplant have introns (Chen et al., 2007a), the cis-acting element(s) responsible for the AM-inducible expression pattern is (are) also expected to be located in the promoter regions of the AM-up-regulated Pht1 genes. The identification of the involved element(s) could greatly facilitate the unravelling of the regulatory mechanism of this transcriptional activation upon AM symbiosis. Recently, several types of cis-regulatory elements involved in transcriptional responses to Pi starvation have been identified by promoter truncation analysis in Arabidopsis and barley (Mukatira et al., 2001; Rubio et al., 2001; Laloi et al., 2004; Schünmann et al., 2004), whereas no experimentally verified elements associated with AM-regulated gene expression have been isolated thus far.

Promoter deletion studies in our work have revealed that Pht1;3, Pht1;4 and Pht1;5 promoters from eggplant and tobacco contain defined regions that are necessary for the expression in colonized cells (Figs 2, 4a). Comparative screening of promoters in homologous genes between more distantly related species (phylogenetic footprinting) have demonstrated that a highly conserved consensus sequence (MYCS) is present in the promoters of AM-regulated PiTs from eudicot species (Fig. 3). The function of MYCS in AM-activated gene expression was further demonstrated by its deletion from both pSmPT4–174 and pNtPT5–199 promoters (Fig. 4b). Targeted deletion of this motif resulted in almost complete absence of GUS activity, suggesting that MYCS is required for the activation of AM-regulated PiT expression. This is also indicative of the presence of a common regulatory pathway, having been conserved over a long period of time and involved in the regulation of the AM Pi-uptake pathway. This hypothesis is well supported by the fact that promoter regions of AM-upregulated StPT3 and MtPT4 directed the expression of the GUS reporter gene in mycorrhizal roots of several eudicot species (Karandashov et al., 2004), and also by the recent study on the petunia mutant, ptd1, which is a regulatory mutant exhibiting normal AM fungal colonization, but repressing all the AM-inducible PiTs, PhPT3-5 in petunia (Wegmüller et al., 2008).

Mycorrhizal Pi uptake might be regulated partially in a P1BS-dependent pathway

It has been commonly recognized that a high P concentration decreases the rate of colonization of plant roots by AM fungi (Amijee et al., 1989; Bruce et al., 1994). It is of interest that colonization still occurred in many plant roots in the presence of a high concentration of P, even though the AM P-uptake pathway might not be functional under such conditions (Nagy et al., 2009). In our previous and present work, we observed that 10–20% of the roots of eggplant, pepper and tobacco were colonized under conditions of high Pi (1 mM); however, the expression of any AM-regulated Pi transporter genes was very weak in the roots (Chen et al., 2007a; Fig 5b). This suggests that the inactivation of AM-induced or AM-up-regulated PiT genes under conditions of high Pi could not be ascribed only to the repression of the symbiosis. The P status of the plant may also act as a direct and important signal in controlling the expression of these genes (by induction and/or suppression).

The promoter-deletion analysis in the present study revealed that MYCS alone was not sufficient to activate the expression of Pht1;3, Pht1;4 and Pht1;5 (Fig. 4b), suggesting the requirement of other cis-regulatory element(s) for the AM-mediated up-regulation of PiT genes. Consequently, further promoter-deletion analysis was performed and underlined another functional short region for each promoter, in which P1BS is the only consensus sequence (Fig. 4a). The importance of P1BS in AM-activated gene expression was further confirmed by its mutation in the promoter region of the NtPT4 gene (Fig. 5a,c). Targeted deletion/mutation of this element resulted in strong reduction or almost complete absence of GUS activity, suggesting that P1BS is also required for maintaining a high level of expression of AM-activated PiT genes.

The P1BS element has been well documented to be a key regulator of expression of many PSI genes by interacting with a Myb transcription factor, PHR1, under conditions of Pi starvation (Daram et al., 1998; Liu et al., 1998; Rubio et al., 2001; Schachtman & Shin, 2007). As P1BS is located near the MYCS element (within 12–94 bp) in the promoter fragments of all AM-regulated PiT genes (Figs 3, 4a), it is tempting to speculate that P1BS and MYCS may be arranged in a combined ensemble that is deciphered by PHR (PHR1 or its homologues) interacting with the other transcription factor(s) recognizing MYCS. Therefore, we propose that there might be two separate, but conserved, regulatory pathways, namely the P1BS/PHR pathway and a MYCS/MYC-transcription factor pathway, which would have become merged in the regulation of AM-activated PiTs in eudicot species. The system could, in turn, explain why conditions of high Pi suppress the expression of the AM-regulated Pht1 genes. However, it should be noted that the PiTs responsible for direct Pi uptake often appear to be down-regulated in roots colonized by AM fungi, suggesting that the regulatory network leading to mycorrhiza/P specificity could well be more subtle and complex. Therefore, whether the two motifs are actually sufficient or still in need of combination with other motifs/transcription factors (positive/negative) for AM-activation remains to be explored further.

Taken together, in the present work we have characterized the two conserved MYCS and P1BS motifs that are involved, at least in solanaceous species, in the regulation of AM-inducible PiTs. We propose that P status is a pivotal regulator controlling the induction/suppression of Pht1 genes at the soil–root interface and also the intraradical symbiotic interface by PHR acting together with other putative protein factors. Given the complexity of the AM Pi-uptake pathway initiated by AM-derived signals, the work presented here offers new insights into the molecular mechanisms underlying the coordination between the AM and the Pi-signalling pathways. Further studies on isolation and subsequent characterization of the transcription factor(s) binding to MYCS and exploring its potential interaction with other transcription factor(s), such as PHR, should be of great significance and would strengthen our understanding on the evolution of response regulation during the establishment of the AM symbiosis.


We thank Dr Lixuan Ren and Mr Guiyun Zhang for technical assistance with AM inoculation. Professor Andrew Smith from the University of Adelaide carefully edited the manuscript revision. We also thank Dr Kin-Ying To from the Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan for kindly providing us with the binary vector pBI121. This work was supported by the 863 program (2006AA10Z134), 973 project (2005CB120903) and National Natural Science Foundation of China.