Functional characterization of the rice SPX-MFS family reveals a key role of OsSPX-MFS1 in controlling phosphate homeostasis in leaves

Authors

  • Chuang Wang,

    1. State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China
    2. Joint Research Laboratory in Genomics and Nutriomics, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China
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    • These authors contributed equally to this work.

  • Wei Huang,

    1. State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China
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    • These authors contributed equally to this work.

  • Yinghui Ying,

    1. State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China
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  • Shuai Li,

    1. State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China
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  • David Secco,

    1. Joint Research Laboratory in Genomics and Nutriomics, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China
    2. Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009, WA, Australia
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  • Steve Tyerman,

    1. Australian Research Council Centre of Excellence in Plant Energy Biology, School of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, PMB1, Glen Osmond, SA 5064, Australia
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  • James Whelan,

    1. Joint Research Laboratory in Genomics and Nutriomics, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China
    2. Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009, WA, Australia
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  • Huixia Shou

    1. State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China
    2. Joint Research Laboratory in Genomics and Nutriomics, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China
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Author for correspondence:
Huixia Shou
Tel: +86 0571 88206133
Email: huixia@zju.edu.cn

Summary

  • Proteins possessing the SPX domain are found in several proteins involved in inorganic phosphate (Pi) transport and signalling in yeast and plants. Although the functions of several SPX-domain protein subfamilies have recently been uncovered, the role of the SPX-MFS subfamily is still unclear.
  • Using quantitative RT-PCR analysis, we studied the regulation of SPX-MFS gene expression by the central regulator, OsPHR2 and Pi starvation. The function of OsSPX-MFS1 in Pi homeostasis was analysed using an OsSPX-MFS1 mutant (mfs1) and osa-miR827 overexpression line (miR827-Oe). Finally, heterologous complementation of a yeast mutant impaired in Pi transporter was used to assess the capacity of OsSPX-MFS1 to transport Pi.
  • Transcript analyses revealed that members of the SPX-MFS family were mainly expressed in the shoots, with OsSPX-MFS1 and OsSPX-MFS3 being suppressed by Pi deficiency, while OsSPX-MFS2 was induced. Mutation in OsSPX-MFS1 (mfs1) and overexpression of the upstream miR827 (miR827-Oe) plants impaired Pi homeostasis in the leaves. In addition, studies in yeast revealed that OsSPX-MFS1 may be involved in Pi transport.
  • The results suggest that OsSPX-MFS1 is a key player in maintaining Pi homeostasis in the leaves, potentially acting as a Pi transporter.

Introduction

Phosphorus (P) is an essential nutrient element for growth and development in all living organisms (Filippelli, 2008). Although P is abundant in the Earth’s crust, it is often present in forms not available to plants. Plants adapt to low inorganic phosphate (Pi) by expansion of the root system to accelerate soil exploration, increasing high-affinity Pi uptake capacity, by adjusting metabolism to maintain intracellular Pi homeostasis, and by induction and secretion of phosphatases and organic acids to mobilize Pi from organic matter (Ticconi & Abel, 2004; Rouached et al., 2010). Intensive research has defined a Pi starvation gene regulatory network that involves various developmental, physiological and metabolic pathways (Schachtman & Shin, 2007; Rouached et al., 2010; Chiou & Lin, 2011). In Arabidopsis, the central regulator of this network is PHR1 and its homologue gene PHR1-LIKE 1 (PHL1), both of which regulate the majority of the Phosphate Starvation Induced (PSI) genes by binding to the PHR1 binding sequences (P1BS, GNATATNC) in the promoters of PSI genes (Rubio et al., 2001; Bustos et al., 2010). Mutation or overexpression of PHR1 changes the expression of a number of PSI genes and the physiological responses to Pi starvation in Arabidopsis (Nilsson et al., 2007; Bustos et al., 2010). In rice, overexpression of OsPHR2– the orthologue of AtPHR1– resulted in changes in root architecture, increased Pi uptake and induction of PSI genes (Zhou et al., 2008; Liu et al., 2010), indicating that a similar regulatory network exists in rice. Several other transcription factors, including WRKY75, ZAT6, bHLH32, MYB62 and WRKY6 in Arabidopsis, and OsPTF1 and OsMYB2P-1 in rice, have also been reported to be involved in Pi starvation responses (Yi et al., 2005; Chen et al., 2007; Devaiah et al., 2007a,b, 2009; Chen et al., 2009; Dai et al., 2012).

The SPX domain (named after proteins SYG1/PHO81/XPR1) is found in several Pi transporters or signalling proteins in yeast and plants(Secco et al., 2012a,b). In yeast, the SPX domain of PHO87 and PHO90, two SPX domain-containing low affinity Pi transporters, has been shown to be required for proper phosphate uptake velocity, phosphate efflux and a phosphate signal transduction, through direct interaction with SPL2 (Hurlimann et al., 2009; Ghillebert et al., 2011; Secco et al., 2012b). In plants, SPX domain-containing proteins can be divided into four classes based on the presence of additional domains in their predicted protein structure (Supporting Information Fig. S1). Class 1 proteins have no other conserved domains except the SPX domain (Duan et al., 2008; Wang et al., 2009) and have already been described as being key players of phosphate homeostasis. In rice the best characterized member of this class, OsSPX1 has been shown to suppress Pi uptake and the phosphate starvation signal pathway via a negative feedback regulation (Wang et al., 2009; Liu et al., 2010). The second class of SPX domain-containing proteins is associated with a C-terminal EXS domain (named after ERD1, XPR1 and SYG1) and constitutes the PHO1 family, involved in phosphate transport and signalling from the root to the shoot in Arabidopsis and rice (Hamburger et al., 2002; Stefanovic et al., 2007; Secco et al., 2010). Class 4 proteins have a RING-type zinc finger domain which may regulate protein degradation (Peng et al., 2007). A recent study in Arabidopsis showed the involvement of one member, AtNLA, in maintaining phosphate homeostasis in a nitrate-dependent manner (Peng et al., 2007; Yaeno & Iba, 2008; Kant et al., 2011). The last subgroup of SPX domain-possessing proteins is the SPX-MFS (Major Facility Superfamily) family. The MFS proteins are the largest group of secondary membrane transporters in the cell, and can function as uniporters, symporters and antiporters with a broad range of substrates (Pao et al., 1998). Interestingly, among the many different MFS families, the phosphate transporter family PHT1 possesses the MFS domain. In rice the SPX-MFS family constitutes four members, with SPX-MFS1 and SPX-MFS2 being the targets of the Pi starvation-induced osa-miR827 (Lin et al., 2010). In addition, in the same study Lin and co-workers, focusing on two members of the family, SPX-MFS1 and SPX-MFS2, demonstrated that they were differentially regulated by Pi starvation and that they were preferentially expressed in the leaves, more precisely in the mesophyll and parenchyma cells surrounding the xylem. However, no function was associated with these two genes. Based on the function of the MFS domain, one interesting hypothesis would be that some of the SPX-MFS family members could be involved in mediating Pi transport and thus controlling shoot Pi homeostasis.

In order to test this hypothesis, we characterized the molecular response of all the members of the SPX-MFS family, as well as the response of osa-miR827 under different Pi conditions. In addition, we confirmed that osa-miR827, and consequently its two target genes SPX-MFS1 and SPX-MFS2, are under the control of the transcription factor OsPHR2. Using the osa-miR827 overexpression and OsSPX-MFS1 mutant plants, we demonstrated that OsSPX-MFS1 is involved in controlling Pi homeostasis in leaves and that OsSPX-MFS1 can transport Pi in yeast.

Materials and Methods

Plant materials and conditions of growth

The Oryza sativa L. japonica variety of Nipponbare was used for all physiological experiments and rice transformations. Hydroponic experiments were conducted using a modified rice culture solution (Yoshida et al., 1976) containing 1.425 mM NH4NO3, 0.323 mM NaH2PO4, 0.513 mM K2SO4, 0.998 mM CaCl2, 1.643 mM MgSO4, 0.25 mM NaSiO3, 0.009 mM MnCl2, 0.075 μM (NH4)6Mo7O24, 0.019 μM H3BO3, 0.155 μM CuSO4 and 0.152 μM ZnSO4 with 0.125 mM EDTA-Fe (II). pH of the solution was adjusted to 5.5. Rice plants were grown in growth chambers with a 12-h photoperiod (200 μmol photons m-2 s−1) and a day : night temperature of 30 : 22°C after germination. Humidity was controlled at 60% RH (relative humidity). For phenotype and Pi concentration measurements, seeds were germinated and grown under HP (10 mg l−1 Pi) or LP (0.1 mg l−1 Pi) conditions for 3 wk.

Construction of vectors and plant transformation

The binary vector pTF101.1 was modified by the introduction of the maize ubiquitin promoter and a nos terminator (Zheng et al., 2010). DNA fragments including the precursor fold-back structure of osa-miR827 gene were amplified from rice genomic DNA. The purified PCR product was inserted into pTF101.1-ubi. The resultant vector was used for Agrobacterium-mediated rice transformation as described previously (Nishimura et al., 2006). The primers used for the amplification are listed in Table S1.

For the GUS construct, the 3.8 kb region upstream ATG of OsSPX-MFS1 was amplified from genomic DNA using primers shown in Table S1. The 5’ primers were extended to add HindIII restrict sites and 3’ primers were extend to add BamHI restrict sites. pBI101.3 was modified in our laboratory by introducing the GUS plus sequence of pCAMBIA1305.1 between BamHI and SacI, and hereafter referred to as pBIGUS-plus. The PCR product of OsSPX-MFS1 promoter was digested by HindIII and BamHI. The purified fragments were introduced into pBIGUS-plus liberalized by HindIII and BamHI. The resultant vector was identified by restriction analysis and sequenced for confirmation. Transgenic lines were made with resulted plasmid by agrobacterium-mediated rice transformation.

Measurement of Pi concentration in plants

Leaves and roots of the WT and transgenic seedlings from either Pi-sufficient or Pi-deficient treatments were sampled separately. The Pi concentration was measured using the procedure described previously (Wang et al., 2009). Briefly, 50 μg of fresh tissue was homogenized with 50 μl of 5M H2SO4 and 3 ml H2O. The homogenate was transferred to 1.5 ml tubes and centrifuged at 10 000 g for 10 min at 4°C. The supernatant was collected and diluted to an appropriate concentration. The diluted supernatant was mixed with a malachite green reagent in 3 : 1 ratio and analysed 20 min afterwards. The absorption values for the solution at 650 nm were determined using a Spectroquant NOVA60 spectrophotometer (Merck, Darmstadt, Germany). Pi concentration was calculated from a standard curve generated with varying concentrations of KH2PO4.

GUS histochemical analysis

Histochemical GUS staining was performed as described previously (Jefferson et al., 1987). In detail, plant tissues harvested from GUS transgenic lines were immediately submerged in GUS staining solution and placed in a vacuum for 10 min. The samples were incubated overnight in darkness at 37°C. Chlorophyll was removed by submerging the tissue in 70% ethanol. Plant material was placed on glass slides and visualized using a stereoscope. The GUS staining solution contained 100 mM sodium phosphate buffer, pH 7.0, Na2EDTA 10 mM, K3[Fe(CN6)] 1 mM, K4[Fe(CN6)] 1 mM, TritonX-100 0.5% (v/v), methanol 20% (v/v), and 0.5 mg ml−1 5-bromo-4chloro-3-indolyl-β-D-glucuronic acid (X-gluc).

Identification of the homozygous mfs1 mutant

The TOS17 insertion mutant of OsSPX-MFS1, mfs1, was obtained from the Rice Genome Resource Center, Japan (RGRC). The homozygous mutant plants were identified using two PCR reactions as instructed at the website of RGRC (http://www.rgrc.dna.affrc.go.jp/index.html). The primers that were used for the two PCR reactions are listed in Table S1.

RNA isolation, cDNA preparation and quantitative real-time PCR

Total RNA was extracted from plant samples using RNeasy mini kits (Qiagen; http://www.qiagen.com) according to the manufacturer’s recommendations. First-strand cDNAs were synthesized from total RNA using SuperScript II reverse transcriptase (Invitrogen). Quantitative real-time PCR was performed using SYBR Premix Ex Taq™ (Perfect Real Time) Kit (TaKaRa Biomedicals, Tokyo, Japan) on a LightCycler480 machine (Roche), according to the manufacturer’s instructions. The amplification program for SYBR Green I was performed at 94°C for 10 s, 58°C for 10 s, and 72°C for 10 s. Triplicate quantitative assays were performed on each cDNA sample. The ACTIN cDNA is an endogenous control that is used to normalize the samples. Relative expression level was calculated by the formula 2−Δ(ΔCp). All the primers that were used for the qRT-PCR are given in Table S1. Three biological experimental repeats were performed for all PCR reactions.

Yeast Pi transport and growth assay

The open reading frame of OsSPX-MFS1 was cloned into yeast expression vector pAG426GPD-ccdB (http://www.addgene.org/14156/). These constructs were transformed into the growth defect of the yeast Pi transport-deficient strain PAM2 (Martinez & Persson, 1998). Transformed yeast was grown in SD(-Ura) medium containing 0.22 mM Pi and 25 mM Na-citrate buffer pH 4.5 to an OD600 of 1. Cells were harvested and washed with water then suspended in fresh medium to an OD600 of 0.2 and incubated at a 30°C shaker. OD600 was monitored as a measurement of growth each 3 h.

Results

Structural analysis of the rice OsSPX-MFS family

In plants, SPX domain-containing proteins are clustered into four groups, based on the presence of additional domains in the C-terminal region. The four families of SPX domain-possessing proteins are designated after their domains, namely the SPX, the SPX-EXS (ERD1, XPR and SYG1), the SPX-RING (Really Interesting New Gene) and the SPX-MFS (Major Facilitator Superfamily) families (Fig. S1). Among the 15 SPX domain-containing proteins found in the rice genome, the SPX-MFS family is composed of four putative genes, referred to as SPX-MFS1 to SPX-MFS4 (Table S2). However, no full length cDNA or EST sequences could be found for SPX-MFS4 (Loc_Os09g34990), therefore the following study only focused on the other three genes, OsSPX-MFS1 (Loc_Os04g48390), OsSPX-MFS2 (Loc_Os02g45520) and OsSPX-MFS3 (Loc_Os06g03860). Protein identity and similarity analysis showed that OsSPX-MFS1 and OsSPX-MFS2 were highly similar with 93.4% and 82.7% of protein identity and similarity, respectively (Fig. 1a). Analysis of the gene structures revealed that the OsSPX-MFS genes have very similar DNA sequence structure with 10 introns and 11 exons including a large intron (> 1.3 kb) in the 5’UTR region (Fig. 1a). The predicted osa-miR827 binding sites are located in the 5’UTR regions of both OsSPX-MFS1 and OsSPX-MFS2 genes, but not in OsSPX-MFS3 (Fig. 1b). Unlike most of the MFS proteins, which possess 12 transmembrane regions, the SPX-MFS proteins have 10 predicted transmembrane domains, with the SPX domain being predicted to be in the opposite cytoplasm space (Fig. 1c).

Figure 1.

Structures of SPX-MFS proteins and SPX-MFS genes. (a) Identity/similarity matrix for OsSPX-MFS proteins. Amino acid similarity and identity are indicated as light blue and pink, respectively. (b) Gene structures of OsSPX-MFS1, OsSPX-MFS2 and OsSPX-MFS3. 5’ and 3’ UTR are shaded yellow, CDS is shaded blue and introns are shaded pink. The miR827 binding site (miR827 BS) is marked in black. Arrows show TOS17 insertion site. UTR, untranslated region; CDS, coding sequence. (c) Predicted topology profile of OsSPX-MFS1 was made and displayed using the TMHMM and Topo2 software (http://www.sacs.ucsf.edu/TOPO2/). SPX domain and MFS domain were highlighted by blue and grey bold amino acids, respectively.

Expression of OsSPX-MFS genes and osa-miR827 in response to Pi starvation

In order to gain insight into the function of the rice SPX-MFS genes, quantitative RT-PCR (qRT-PCR) was performed on RNA samples extracted from 3-wk-old rice seedlings grown at different Pi conditions. Under Pi-sufficient conditions, transcript analysis showed that the three members of the SPX-MFS family were expressed mainly in the leaves (Fig. S2). Furthermore, phosphate availability in the media differentially regulated the expression of the SPX-MFS transcripts. While the expression of SPX-MFS1 was already low at 5 mg l−1 Pi, and decreased further at lower Pi concentration, the expression of OsSPX-MFS3 was only suppressed below 0.5 mg l−1 Pi supply (Fig. 2a). By contrast, the transcripts of OsSPX-MFS2 and osa-miR827 were greatly induced by Pi starvation (Fig. 2a). Similar responses to Pi starvation were observed in the roots (Fig. S3A). To further characterize the effect of Pi starvation on the expression of the members of the SPX-MFS genes, a time course experiment was performed as follows: 10-d-old seedlings grown under sufficient Pi condition were transferred to Pi-deficient conditions for 1, 2, 3, 5, 7 and 10 d, before being re-supplied with Pi for 1 d. The three transcripts of SPX-MFS genes showed a slow response to Pi starvation, reaching their maximum or minimum levels of expression after 10 d of Pi starvation (Fig. 2b). Interestingly, the expression levels of SPX-MFS2, SPX-MFS3 and osa-miR827, but not SPX-MFS1, were quickly reversed after 1 d of Pi re-supply (Fig. 2b). Similar patterns of expression were observed in the roots (Fig. S3B).

Figure 2.

Expression pattern of OsSPX-MFS1, OsSPX-MFS2, OsSPX-MFS3 and osa-miR827 in the leaves in response to phosphate (Pi) starvation. (a) Effects of varying Pi concentrations on the expression of SPX-MFS and osa-miR827 transcripts. Rice (Oryza sativa) seeds were geminated and grown on nutrient solutions with different Pi concentrations for 3 wk. Leaves were sampled separately for analysis. (b) Effects of Pi starvation duration on the expression of SPX-MFS and osa-miR827 transcripts. Seeds were geminated and grown on full nutrient solution (10 mg l−1 Pi) for 10 d before being transferred to Pi-deficient conditions (0.1 mg l−1, T = 0 d) for the desired period of time (from 0 to 10 d). Leaves were sampled at different time following treatment (from 0 to 10 d). After 10 d of Pi starvation, plants were re-supplied with Pi for 1 d. All data are means of three biological replicates with error bars indicating SD. CK, Pi sufficient condition; R1d, 10 d Pi starvation followed by 1 d Pi sufficient supplement. Expression of OsACTIN was used as the internal control.

OsPHR2 regulates the expression of osa-miR827, OsSPX-MFS1 and OsSPX-MFS2

In order to determine if the osa-miR827-SPX-MFS1/2 pathway is under the control of OsPHR2, the central transcription factor positively regulating Pi homeostasis in rice (Rubio et al., 2001; Zhou et al., 2008; Bustos et al., 2010), we compared the expression levels of osa-miR827, OsSPX-MFS1, OsSPX-MFS2 and OsSPX-MFS3 using the previously reported OsPHR2 overexpression plants (Zhou et al., 2008). Analysis of the promoter region of osa-miR827 showed the presence of the P1BScis-elements, the binding site of OsPHR2 (Fig. S4). Under sufficient Pi conditions, the transcript abundance of pri-miR827 was increased eight-fold in OsPHR2 overexpression plants compared to WT plants, in the leaves (Fig. 3). Accordingly, the targets of osa-miR827, OsSPX-MFS1 and OsSPX-MFS2, were downregulated in the OsPHR2 overexpression plants compared to WT (Fig. 3). The expression OsSPX-MFS3, which is not a target of osa-miR827, was unaffected by the overexpression of OsPHR2.

Figure 3.

Expression pattern of Pri-miR827, OsSPX-MFS1, OsSPX-MFS2 and OsSPX-MFS3 in WT (open bars) and OsPHR2 overexpression (closed bars) plants. Rice (Oryza sativa) seeds were geminated and grown on full nutrient solution for 3 wk. Total RNA was extracted from the leaves and used for qRT-PCR. All data are means of three biological replicates with error bars indicating SD. Expression of OsACTIN was used as the internal control.

Mutation in OsSPX-MFS1 alters Pi re-mobilization in rice

In order to investigate the function of the SPX-MFS family, we searched different rice genomic databases for publicly available mutants. While no mutants could be found for OsSPX-MFS2 and OsSPX-MFS3, a TOS17 transposon insertion mutant of OsSPX-MFS1, mfs1, was found, with the transposon being inserted in the first intron of the gene (Fig. 1a). Homozygous mutants were identified by PCR (data not shown). Analysis by qRT-PCR showed that the mfs1 is a knock-down mutant, with decreased SPX-MFS1 transcript levels compared to WT, both under Pi-sufficient and -deficient conditions (Fig. 4a). However, the expression levels of OsSPX-MFS2 and OsSPX-MFS3 were unaffected by the mutation in SPX-MFS1 (Figs 4, S4). In addition to the mfs1 mutant, transgenic plants overexpressing osa-miR827 were generated for the study. DNA fragments including the precursor fold-back structure of osa-miR827 were amplified and inserted into the binary vector pTF101.1 under the control of a maize Ubiquitin-1 promoter. The resulting construct was used for rice transformation. Among the different osa-miR827 overexpressing lines obtained, three lines –osa-miR827-Oe1, osa-miR827-Oe2 and osa-miR827-Oe3 – were analysed and showed increased pri-miR827 transcript levels compared to WT under both Pi-sufficient and -deficient conditions (Fig. 4d). Overexpression of osa-miR827 greatly decreased the expression of OsSPX-MFS1 under both Pi-sufficient and -deficient conditions, and that of OsSPX-MFS2 under Pi-deficient conditions (Fig. 4a,b), which is consistent with reported results (Lin et al., 2010). The expression of OsSPX-MFS3 was not influenced by the alteration of osa-miR827. These expression patterns were similar in both leaves and roots under all the tested conditions (Fig. S5).

Figure 4.

Expression analysis ofOsSPX-MFS1 (a), OsSPX-MFS2 (b), OsSPX-MFS3 (c) and Pri-miR827 (d) in osa-miR827 overexpression lines and msf1. Rice (Oryza sativa) seeds were geminated and grown on nutrient solutions with high phosphate (Pi) concentration (HP, open bars; 10 mg l−1) and low Pi concentration (LP, closed bars; 0.1 mg l−1) for 3 wk. Total RNA was extracted from the leaves and used for qRT-PCR. All data are means of three biological replicates with error bars indicating SD. Expression of OsACTIN was used as the internal control. WT, wild- type; Oe1/2/3, osa-miR827 overexpression lines; mfs1, OsSPX-MFS1 mutant line NC2634.

The Pi concentrations of roots and shoots were assessed in plants grown in Pi-sufficient (10 mg Pi l−1; HP, high phosphorus) and -deficient (0.1 mg Pi l−1; LP, low phosphorus) conditions for 3 wk. Interestingly, Pi concentration in leaves of the miR827-Oe lines and mfs1 mutant was significantly higher than that of the WT plants (Fig. 5a,b). In the roots, Pi concentration was not significantly different between wild-type (WT) and transgenic mutant plants under HP and LP conditions (Fig. 5a,b).

Figure 5.

(a) Phosphate (Pi) concentration of the second leaves and roots in 3-wk-old osa-miR827 overexpression and OsSPX-MFS1 mutant rice (Oryza sativa) plants grown on nutrient solution with high Pi concentration (HP, 10 mg l−1). (b) Pi concentration of the second leaves and roots in 3-wk-old osa-miR827 overexpression and mfs1 mutant plants grown on nutrient solution with low Pi concentration (LP, 0.1 mg l−1) for 3 wk. (c) Pi concentration in different leaves of the WT, osa-miR827 overexpression and mfs1 mutant plants. Seeds were geminated and grown on HP or LP nutrient solutions for half a month. Individual leaves were sampled for measurements. All data are means of three biological replicates with error bars indicating SD. Significant differences are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001. WT, wild- type; Oe1/2/3, osa-miR827 overexpression lines; mfs1, OsSPX-MFS1 mutant line NC2634.

We further examined the involvement of SPX-MFS1 in regulating Pi homeostasis in the leaves by analysing Pi concentration in individual leaves of WT, osa-miR827 overexpression and mfs1 plants grown for 2 wk under Pi-sufficient conditions. At this age, the seedlings have five leaves. The Pi concentrations of the second to fifth leaves were measured. As already observed, the Pi concentration in the second leaf of mfs1 was higher than that in the second leaf of WT plants, with more than four-fold difference (Fig. 5c). Similar differences were observed in all the old leaves, with Pi concentration being more than thrice and twice as high as WT in the third and fourth leaves of mfs1, respectively (Fig. 5c). Interestingly, in the youngest leaf, no significant differences were observed in Pi concentration. A similar phenomenon was observed in the osa-miR827 overexpression plants also, although to a lesser extent.

Spatial expression of OsSPX-MFS1

As changed expression of OsSPX-MFS1 altered Pi homeostasis, we studied the expression pattern of OsSPX-MFS1 in the various tissues. A 3.8 kb promoter region upstream ATG of OsSPX-MFS1 gene, which includes 5’UTR and the first intron, was amplified and fused to GUS reporter gene to make an OsSPX-MFS1:GUS construct. As expected, the transgenic plants showed strong staining in all the leaves tested and weak staining in the roots under Pi-sufficient conditions (Fig. 6a,e). Interestingly, we also observed a high expression of OsSPX-MFS1 in the base of leaf sheath (Fig. 6b,d), collar (Fig. 6c) and root junction (Fig. 6e), indicating a function of OsSPX-MFS1 in these tissues.

Figure 6.

Spatial expression of GUS reporter gene under the control of the OsSPX-MFS1 promoter. (a) Rice (Oryza sativa) leaf; (b) stem with leaf sheath; (c) leaf collar (red arrowhead); (d) base leaf sheath; (e) roots (red arrowhead shows root junction). Bars: (a–d) 2 mm; (e) 1 cm.

Effect of the mutation of OsSPX-MFS1 on phosphate signalling

In order to examine whether or not OsSPX-MFS1 played a role in Pi starvation signalling, the transcript abundance of several root PSI genes was determined. Upon Pi starvation, all the tested PSI genes, including OsSPX1, OsPAP10a, OsIPS1, OsACP1, OsPT2, OsPT3, OsPT7 and OsPT8 were upregulated in WT, osa-miR827 overexpression and mfs1 plants (data not show). However, the extent of induction of the PSI genes by Pi starvation was attenuated in the osa-miR827 overexpression and mfs1 plants (Fig. 7). Indeed, under Pi-deficient conditions, genes such as IPS1 and PT3, were five and three times less induced in the mfs1 mutant than in the WT plants, respectively.

Figure 7.

Expression of phosphate (Pi) starvation-induced genes in mfs1 mutant plants under low-Pi (0.1 mg l−1) conditions. Rice (Oryza sativa) seeds were germinated and grown on nutrient solutions with low Pi concentration (0.1 mg l−1) for 3 wk. Total RNA was extracted from the roots and used for qRT-PCR. All data are means of three biological replicates with error bars indicating SD. Expression of OsACTIN was used as the internal control.

Complementation of a yeast Pi-transport mutant

In order to test the possibility that OsSPX-MFS1 could function as a Pi transporter, a functional complementation approach of a yeast strain (PAM2) lacking the two high-affinity Pi uptake transporters was undertaken (Martinez & Persson, 1998). To do so, the full length cDNA sequence of OsSPX-MFS1 was amplified by RT-PCR and cloned into a yeast expression vector, before being transformed in the PAM2 yeast strain. The growth rate of cells expressing OsSPX-MFS1 as well as the empty vector was monitored under low external Pi, in a pH-specific manner. While no significant differences of growth rate between cells expressing the empty vector or OsSPX-MFS1 could be observed at pH 6 and pH 7, the cells expressing OsSPX-MFS1 had a significantly higher growth rate at pH 4 and pH 5, compared to the one expressing the empty vector (Fig. 8).

Figure 8.

Complementation of a yeast high-affinity phosphate (Pi) transport mutant by OsSPX-MF1. Yeast PAM2 cells harbouring either an empty expression vector (control; triangles) or OsSPX-MF1 (circles) gene constructs were grown in synthetic dextrose -Ura medium to the logarithmic phase, OD600 ≈ 1.0 and then suspended to OD600 = 0.2 in fresh SD medium containing 0.22 mM Pi and 25 mM Na-Citrate buffer with indicated pH, incubated at 30°C. OD600 was monitored as a measurement of growth.

Discussion

The SPX domain is found at the N-terminal region in a variety of proteins often involved in Pi signalling or transport (Secco et al., 2012a,b). In this work, we studied the involvement of the rice SPX-MFS family in phosphate homeostasis. The rice genome has three validated genes possessing both the SPX and MFS domains. Proteins containing the MFS domain are the largest group of secondary membrane transporters, transporting small solutes, including sugar and Pi (Pao et al., 1998; Abramson et al., 2004; Lemieux et al., 2004). Interestingly, the structure of SPX-MFS proteins is very similar to the three yeast low-affinity Pi transporters, namely PHO87, PHO90 and PHO91, containing a C-terminal transporter activity domain and an N-terminal SPX domain (Hurlimann et al., 2007, 2009). The SPX domain in the three yeast low-affinity Pi transporters is proposed to act as an external Pi sensor, thereby regulating Pi starvation signalling responses in yeast (Pinson et al., 2004). Under Pi-deficient conditions, SPL2 (Suppress the temperature-sensitive phenotype of the plc1Δ2) downregulates the transport activity of PHO87 and PHO90 by direct physical interaction via the SPX domain (Hurlimann et al., 2009). Reduction of the low-affinity Pi transporter activity results in a decrease of internal phosphate content, which induces the central transcription factor PHO4 and its downstream high-affinity Pi transporters PHO84 (Wykoff et al., 2007). In this study, we show that OsSPX-MFS1 can complement the growth defect of the PAM2 yeast strains, depleted of both high-affinity Pi uptake transporters, suggesting that OsSPX-MFS1 may have Pi transporter activity. In addition, this activity appears to be pH dependent, having an optimum activity at pH 4.0. Consistently, mutation of OsSPX-MFS1 or overexpression of osa-miR827 resulted in impaired Pi homeostasis in the leaves, with older leaves overaccumulating Pi. These results contradict those from Lin et al. (2010) where no phenotype could be detected with overexpression of osa-miR827. However, in our study Pi concentrations of individual leaves were measured, enabling the identification of subtle changes in Pi concentration, which may not be detected when assessing whole shoot Pi concentration as in the case of Lin et al. (2010). In addition, the osa-miR827 overexpressing plants have a weaker phenotype than the mfs1 mutant, accumulating intermediate concentrations of Pi in old leaves, potentially explaining the lack of phenotype of the osa-miR827 overexpressing lines reported by Lin et al. (2010).

Several miRNAs, such as miR399, miR447, miR778, miR827 and miR2111, have already been demonstrated to be induced upon Pi starvation (Pant et al., 2009; Hsieh et al., 2009). Among them, miR399 has been the most intensively studied, revealing that the miR399-PHO2 pathway is conserved in both Arabidopsis and rice, and is under the control of PHR, the central transcription factor maintaining Pi homeostasis (Bari et al., 2006; Liu et al., 2010). miR827 exists in numbers of plant species and has already been demonstrated to be specifically induced by Pi starvation (Lu et al., 2008; Hsieh et al., 2009; Pang et al., 2009; Xue et al., 2009; Zhang et al., 2009). Our work, as well as the study of Lin et al. (2010), showed that the rice osa-miR827 is preferentially expressed in the leaves, is greatly induced by Pi starvation and regulates the expression of OsSPX-MFS1 and OsSPX-MFS2, and that they are all expressed in the same cells, mainly around the vascular bundles and in parenchyma cells associated with xylem (Lin et al., 2010). Furthermore, this pathway, similarly to the miR399-PHO2 pathway, is under the control of the central transcription factor OsPHR2. The Arabidopsis orthologue of osa-miR827, ath-miR827, is also upregulated by Pi starvation, but is preferentially expressed in the roots. In addition ath-miR827 was shown to target a single gene, AtNLA, recently described to be involved in regulating Pi homeostasis in a nitrate-dependent manner, by regulating the expression of a high-affinity Pi transporter PHT1.1 and its traffic facilitator, PHF1 (Kant et al., 2011). Indeed, AtNLA is a member of the SPX-RING family, and encodes E3 ubiquitin ligase, involved in protein degradation. In Arabidopsis, the miR399-PHO2 and miR827-NLA pathways are thought to be coordinated in order to allow fine-tuning of the expression of PHT1.1 and PHF1 (Kant et al., 2011). Therefore, ath-miR827 and osa-miR827, via NLA1 and SPX-MFS1, are involved in regulating leaf Pi homeostasis, by negatively regulating the expression of different Pi transporters.

Under Pi-limiting conditions, overexpression of osa-miR827 or mutation in OsSPX-MFS1, despite having similar Pi concentrations as WT plants in roots, showed a reduction in the level of induction of some PSI genes in roots, compared to WT. Because OsSPX-MFS1 may function as a transporter, it is unlikely that OsSPX-MFS1 directly regulates expression of PSI genes. Rather, the reduction of induction observed for some PSI genes, could be explained by higher Pi concentration in leaves detected in the mfs1 mutants and osa-miR827 overexpressing plants, compared to WT. It is suggested that OsSPX-MFS proteins may localize on tonoplast and regulate Pi storage in parenchyma cells. Under Pi starvation conditions, the cells will mobilize Pi stored in vacuoles, which will attenuate the phosphate starvation response of the cell (Pratt et al., 2009). Thus, reducing the level of expression of OsSPX-MFS1 may increase the Pi concentration in cytoplasm, which in turn could suppress the expression of PSI genes. Interestingly, while the residual level of expression of OsSPX-MFS1 is higher in the mfs1 mutants than the osa-miR827 overexpressing plants, the Pi concentration in old leaves is higher in the mfs1 mutants than the osa-miR827 overexpressing plants. In addition, the level of expression of OsSPX-MFS2 is downregulated in the osa-miR827 overexpressing lines, compared to WT and mfs1. Thus, it is tempting to hypothesize that OsSPX-MFS1 and OsSPX-MFS2 could have opposite functions, fine-tuning Pi concentration in leaves. This hypothesis is also strengthened by the expression profiles of OsSPX-MFS1 and OsSPX-MFS2, being oppositely regulated by Pi availability. In addition OsSPX-MFS proteins are predicted to be localized on the tonoplast, thus OsSPX-MFS members could be involved in regulating vacuolar Pi concentration. Indeed in higher plants, vacuoles are thought to have dual roles as both sink and source for Pi, making them a key component in buffering Pi fluctuations due to external Pi supply or metabolic changes.

In summary, under Pi starvation, OsSPX-MFS1 and OsSXP-MFS2 are negatively regulated by the osa-miR827/OsPHR2 pathway. Mutation of OsSPX-MFS1 resulted in disturbed Pi homeostasis in the leaves, associated with altered responses of some of the PSI genes. Heterologous complementation studies also revealed that OsSPX-MFS1 could mediate Pi transport in yeast.

Acknowledgements

This work was supported by the Key Basic Research Special Foundation of China (2011CB100303), National Science Foundation of China (31172024), the Ministry of Science and Technology of China (20080242 and 2011ZX08004-001-03), Zhejiang Bureau of Science and Technology (R3090229), Harvestplus-China (8237) and the Australian Research Council Super Science Fellowship (FS100100022). The authors thank the RGRC (Rice Genome Resource Center, Japan) for the TOS17 insertion mutants NC2634.

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