Arbuscular mycorrhizal (AM) fungi accumulate a massive amount of phosphate as polyphosphate to deliver to the host, but the underlying physiological and molecular mechanisms have yet to be elucidated. In the present study, the dynamics of cationic components during polyphosphate accumulation were investigated in conjunction with transcriptome analysis.
Rhizophagus sp. HR1 was grown with Lotus japonicus under phosphorus-deficient conditions, and extraradical mycelia were harvested after phosphate application at prescribed intervals. Levels of polyphosphate, inorganic cations and amino acids were measured, and RNA-Seq was performed on the Illumina platform.
Phosphate application triggered not only polyphosphate accumulation but also near-synchronous and near-equivalent uptake of Na+, K+, Ca2+ and Mg2+, whereas no distinct changes in the levels of amino acids were observed. During polyphosphate accumulation, the genes responsible for mineral uptake, phosphate and nitrogen metabolism and the maintenance of cellular homeostasis were up-regulated.
The results suggest that inorganic cations play a major role in neutralizing the negative charge of polyphosphate, and these processes are achieved by the orchestrated regulation of gene expression. Our findings provide, for the first time, a global picture of the cellular response to increased phosphate availability, which is the initial process of nutrient delivery in the associations.
Arbuscular mycorrhizal (AM) fungi form symbiotic associations with most land plants and promote the growth of host plants through enhanced uptake of mineral nutrients, particularly phosphorus (P; Smith & Read, 2008). The fungi take up inorganic phosphate (Pi) from the soil and accumulate it as polyphosphate (polyP), which is probably involved in the long-distance translocation of Pi through hyphae (Hijikata et al., 2010). PolyP is a linear chain of three to hundreds of Pi residues linked by high-energy phosphoanhydride bonds (Kornberg et al., 1999). AM fungi grown under P-deficient conditions are capable of accumulating a massive amount of polyP within several hours in response to increased Pi availability (Hijikata et al., 2010), which is considered to be an adaptive trait commonly evolved in microorganisms to prepare for P deficiency, namely ‘polyP overcompensation (overplus)’ (Harold, 1966). In natural ecosystems, patches of nutrients are associated with decomposing organic residues, for example, leaf litter and dead bodies of animals (reviewed in Tibbett, 2000). Therefore, polyP overplus is an important trait that allows AM fungi to capture Pi efficiently from such nutrient patches, which consequently has a large impact on the nutrient acquisition strategies of plants.
Fungi take up Pi mainly through the high-affinity H+/Pi symporter, which was first identified in Saccharomyces cerevisiae as PHO84 (Bun-Ya et al., 1991). The symporter also plays a significant role in Pi uptake in AM fungi (Harrison & van Buuren, 1995). In contrast with extensive studies on the H+/Pi symporters, the enzyme responsible for polyP biosynthesis in eukaryotic microorganisms has only recently been identified in S. cerevisiae; a complex of vacuolar transporter chaperones (VTC complex) at the vacuolar membrane polymerizes the γ-phosphate of ATP supplied in the cytosol and releases the polymer into the vacuole (Hothorn et al., 2009). It is highly likely that AM fungi also synthesize polyP via the VTC complex (Tisserant et al., 2012) using ATP as substrate (Tani et al., 2009) (Fig. 1). In the newly released genome sequence of the model AM fungus Rhizophagus irregularis, c. 23 000 genes were predicted and expressed in spores and/or intraradical mycelia (Tisserant et al., 2013). Transcriptional regulation of the genes in response to environmental changes, however, has yet to be elucidated. In particular, information about responses to increased Pi availability in extraradical mycelia is of great interest, but still fragmented.
PolyP is a polyanionic compound, and fully dissociated polyP has one negative charge per Pi residue in addition to the two extra charges of terminal residues (Supporting Information Fig. S1). PolyP overplus, therefore, should result in the accumulation of a large amount of negative charge in the cell, suggesting that there would be a regulatory mechanism for maintaining charge neutrality of the cell. Inorganic cations and basic (cationic) amino acids are likely to be potential counter ions for polyP (Fig. 1). In four ectomycorrhizal fungi (Bücking & Heyser, 1999) and an AM fungus (Bücking & Shachar-Hill, 2005), energy-dispersed X-ray spectroscopic analysis revealed that various di- and monovalent cations, magnesium (Mg2+), calcium (Ca2+), potassium (K+) and sodium (Na+), were co-localized with P in their vacuoles. In Neurospora crassa, these four cations were also detected in the isolated vacuoles in which polyP was accumulated (Cramer & Davis, 1984). On the other hand, arginine, which has one basic side group in the molecule, is the most abundant amino acid in AM fungi (Johansen et al., 1996; Jin et al., 2005) and a translocation form of nitrogen towards the plants (Govindarajulu et al., 2005), leading to the idea that arginine may be associated with polyP and co-translocated (Bago et al., 2001). In vivo ionic interactions between basic amino acid and polyP, however, are controversial; polyP degradation results in the release of an equivalent amount of arginine from the vacuole in S. cerevisiae (Dürr et al., 1979), whereas, in N. crassa, basic amino acids are retained in the vacuoles even after most polyP has been degraded (Cramer & Davis, 1984). Accordingly, the elucidation of the quantitative relationship between polyP and these cationic components is necessary to understand the regulatory mechanism of charge neutrality during polyP accumulation.
RNA-Seq, the next-generation sequencing technology for transcriptome analysis, provides a largely unbiased method to explore comprehensive gene structure and expression profiles. Moreover, we have developed a mass production system for extraradical mycelia of AM fungi, which has enabled us to study polyP biosynthesis (Tani et al., 2009) and dynamics (Hijikata et al., 2010). In the present study, the mass production system was applied for the quantitative analysis of cationic components, and also for the preparation of high-quality RNA for RNA-Seq, to obtain a comprehensive understanding of the cellular and molecular processes involved in polyP overplus in AM fungi.
Materials and Methods
The AM fungus Rhizophagus sp. strain HR1 (MAFF520076) was maintained with sand culture in a glasshouse, and GenBank accession numbers of the small- and large-subunit ribosomal RNA gene (SSU rDNA and LSU rDNA) sequences are AB220171 and AB370889, respectively. The SSU rDNA sequence shows high similarity to those of R. manihotis (Y17648), R. clarus (AJ852597) and R. intraradices (FR750209 and EU232660), whereas the LSU rDNA sequence shows high similarity to those of R. intraradices (DQ469108 and FJ235569) and R. irregularis (FR750084 and FR750070). This strain produces many hyphae with few spores during vegetative growth of the host plant, and thus is suitable for physiological study.
Lotus japonicus L. cv Miyakojima MG-20 (National Bioresource Project Legume Base, http://www.legumebase.brc.miyazaki-u.ac.jp/top.jsp) was sown on moistened filter paper in a Petri dish and germinated at 25°C for 2 d in the dark. Four seedlings were transplanted to the mycorrhizal compartment (MC) of the mesh bag culture system in a 120-ml plastic pot (6 cm in diameter) and inoculated with Rhizophagus sp. HR1 at 500 spores per pot. The mesh bag system consisted of the MC and a hyphal compartment (HC), which were separated by a cone-shaped 37-μm nylon mesh bag (Nippon Rikagaku Kikai, Tokyo) with a volume of 26 ml (Fig. S2). The pore size of the nylon mesh was sufficiently small to prevent L. japonicus roots from passing through, but large enough to allow AM fungal hyphae to pass through. In a previous study (Hijikata et al., 2010), a P diffusion barrier between the compartments was inserted, but not in the present study, because the diffusion of a minimum amount of Pi from HC to MC across the nylon mesh did not affect polyP accumulation in extraradical mycelia in HC, at least within 24 h (data not shown). Media in the two compartments were prepared as follows: river sand was washed with tap water by decantation, dried, separated by stainless steel sieves into two fractions (particle size distributions of 0.1–3 mm and 1–3 mm for MC and HC, respectively), rinsed with deionized water (DIW) and autoclaved. The sand in HC (1–3 mm) enabled rapid and efficient separation of mycelia from sand particles, which was essential for elemental analysis and the preparation of high-quality RNA, whereas that in MC (0.1–3 mm) was optimized for plant growth. The seedlings were grown in a growth chamber (16-h photoperiod and 25°C with a photosynthetic photon flux density of 150 μmol m−2 s−1) and thinned to two plants per pot after 1 wk. The plants received DIW every other day for the first week, low-P nutrient solution (4 mM NH4NO3, 1 mM K2SO4, 0.75 mM MgSO4, 2 mM CaCl2, 50 μM Fe-Na-EDTA, 50 μM KH2PO4, pH 5.4) for the second to sixth week, and non-P nutrient solution (KH2PO4 was withheld from the low-P nutrient solution) for the seventh week in a sufficient amount until the solution flowed out from the drain. At the beginning of the eighth week, a 1 mM KH2PO4 (pH 4.8) solution was applied to HC using a pipette in a sufficient amount until the solution flowed out from the drain (c. 15 ml). The Pi solution was washed out with DIW in a sufficient amount 1 h after Pi application, and extraradical mycelia in HC were harvested from two pots together by wet sieving at prescribed time intervals and combined as one sample. Immediately after harvest, all visible sand particles adhering to the mycelia were removed under a dissecting microscope with forceps as quickly as possible, and the mycelia were frozen in liquid nitrogen and stored at −80°C. At all time points, six pots were harvested to obtain three replicates (n =3).
PolyP, inorganic cation and amino acid analyses
The frozen mycelial sample (c. 20–40 mg fresh weight (FW) per sample) was ground on an ice-cooled mortar and pestle with a 30-fold volume (v/w) of 10 mM Tris-HCl buffer (pH 8.0). Subsamples for protein (10 μl) and amino acid (200 μl) analyses were taken from the slurry and stored at −30°C. Then, 300 μl of the slurry were mixed with 219 mg of powdered urea (the final concentration of urea was 8 M) and centrifuged at 18 000 g for 10 min at 10°C. For inorganic cation analysis, 250 μl of the supernatant were taken and stored at −30°C. For polyP analysis, 75 μl of the supernatant were desalted immediately after centrifugation with a Micro Bio-Spin P-6 gel filtration spin column (Bio-Rad Laboratories) pretreated with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and the polyP concentration was determined by the reverse reaction of polyP kinase (PPK) purified from PPK-overexpressing Escherichia coli (Ault-Riché et al., 1998), as described previously (Ezawa et al., 2003). The concentration of polyP was expressed in terms of molar electric charge (molc) of Pi residues per unit protein. In this calculation, one negative charge per residue was assumed, that is a polyPn molecule possesses n charges (where n represents the number of Pi residues), although a fully dissociated polyPn molecule theoretically possesses n + 2 charges, the two extra charges of which are the terminal residues at both ends. This is because (i) the terminal charges might be negligible if the chain length of polyP in AM fungi is more than 300, as estimated in Ezawa et al. (2003) and (ii) accurate estimation of the chain length distribution of polyP is technically difficult. For inorganic cation analysis, the stored supernatant (250 μl) was heated in 2.0 ml of 15% (v/v) sulfuric acid at 105°C for 1 h to evaporate water (until sulfuric acid was concentrated) and then digested at 105°C for 5 h by adding 0.1 ml hydrogen peroxide four times at 1-h intervals as an oxidant. Concentrations of sodium (Na+), potassium (K+), calcium (Ca2+), magnesium (Mg2+), ferric (Fe2+), zinc (Zn2+), aluminum (Al3+), copper (Cu2+), nickel (Ni2+) and manganese (Mn2+) ions in the digests were determined by an inductively coupled plasma (ICP) mass spectrometer (ELAN DRC II; PerkinElmer, Yokohama, Japan) using the ICP multi-element standard solution IV (Merck, Tokyo) as standard. Concentrations of the cations were given in terms of molar electric charge (molc) per unit protein. For free amino acid analysis, the slurry (200 μl) was mixed with an equal volume of 20% (w/v) trichloroacetic acid, kept for 2 h at 4°C, and centrifuged at 18 000 g for 15 min at 4°C; then trichloroacetic acid was extracted three times with ice-cooled 500 μl water-saturated dimethyl ether. After air drying for 1 h, 160 μl of the solution were mixed with 40 μl of 0.1 M hydrochloric acid, filtered through a 0.45-μm Millex-LH polytetrafluoroethylene (PTFE) membrane filter (Millipore) and introduced to an amino acid analyzer (L-8500; Hitachi, Tokyo). Protein concentration was determined with a DC Protein Assay Kit (Bio-Rad Laboratories) according to the manufacturer's instructions using bovine serum albumin as standard. For the conversion of concentration data (per unit protein) to those on the basis of unit dry weight (DW), or vice versa, the conversion coefficients 13–18 mg protein g−1 FW with 80% (w/w) water content, which were determined using several lots of mycelia, were used. StatView software (SAS Institute Inc., Cary, NC) was employed for analysis of variance (ANOVA) and Tukey–Kramer post-hoc test.
RNA-Seq and expression analysis
Total RNA was extracted from the frozen mycelia (20–40 mg FW) obtained at time zero and 4 h after Pi application with an RNeasy Plant Mini Kit (Qiagen), followed by RNase-free DNase I (Qiagen) treatment (n =3 for each time point). Sequencing libraries were constructed using 500 ng of total RNA with a TruSeq RNA Sample Prep kit (Illumina, Tokyo) following the manufacturer's instructions. Paired-end 101-bp × 2 sequencing was performed with HiSeq2000 (Illumina). Raw sequence data were deposited in the DDBJ Sequence Read Archive under accession number DRA001877. High-quality reads for which > 90% bases showed Phred quality scores > 20 were extracted and assembled with Trinity (Grabherr et al., 2011).
The assembled sequences (contigs) were queried against the predicted gene models (Gloin1_all_transcripts_20120510.nt.fasta) and expression sequence tags (ESTs) (Gloin1_ESTs_20120510_Combest_RNA_and_EST_contigs.fasta) of R. irregularis DAOM 181602 in the DOE Joint Genome Institute (http://genome.jgi.doe.gov/Gloin1/Gloin1.home.html), as well as against the GenBank nucleotide collection with the BLASTN algorithm at an e-value cutoff of 10−5. Contigs that showed higher similarity (i.e. a lower e-value and a higher bit score) to the sequences in the R. irregularis genome database than those in GenBank were defined to be of Rhizophagus sp. HR1 origin. Open reading frames (ORFs) of 50 or longer amino acid residues were predicted and extracted from the contigs using the utility program of Trinity (transcripts_to_best_scoring_ORFs.pl) and clustered using CD-HIT-EST with a 95% sequence identity cutoff. The longest ORFs were selected as representatives from each cluster to minimize duplication of putative splice variants, polymorphisms and fragmented (frameshifted) ORFs in the predicted gene set (Li & Godzik, 2006). Putative functions of the ORFs were assigned with reference to the S. cerevisiae protein database (orf_trans.fasta) in the Saccharomyces Genome Database (SGD) (http://www.yeastgenome.org/) and the N. crassa protein database (neurospora_crassa_or74a__finished__10_proteins.fasta) at the Broad Institute (http://www.broadinstitute.org/annotation/genome/neurospora/MultiHome.html) by the BLASTP algorithm at an e-value cutoff of 10−5. The gene ontology (GO) functional classes were assigned with reference to those of S. cerevisiae (go_terms.tab) in SGD and N. crassa (go_for_nc12.tsv) at the Broad Institute.
Expression levels of transcripts were estimated on the basis of the number of reads that were uniquely mapped to the corresponding ORF sequences using the Burrows–Wheeler Aligner's Smith–Waterman algorithm with default parameters, in which five nucleotide mismatches were allowed in the alignment between a read (101 bp) and the ORF sequence (Li & Durbin, 2010). The reads that were mapped to multiple locations in an ORF or to multiple ORFs were discarded using SAMtools (Li et al., 2009). The raw read counts were normalized with the iDEGES/edgeR method (Sun et al., 2013), and pairwise comparisons of digital gene expression between the time points were conducted with the edgeR program in R (Robinson et al., 2010). The false discovery rate (FDR) adjustments (Benjamini & Hochberg, 1995) were applied to correct for multiple testing, and differentially expressed genes were identified with an FDR threshold of 0.05. To identify GO terms in which up- or down-regulated genes were over-represented, the enrichment analysis tool in the Blast2GO program (Conesa et al., 2005) was employed with an FDR threshold of 0.05.
Total RNA (500 ng) was reverse transcribed with the mixture of random and oligo dT primers using a PrimeScript RT Master Mix Kit (Takara, Ohtsu, Shiga, Japan), and quantitative PCR was performed using SYBR Premix Ex Taq II (Takara) with LightCycler (Roche), according to the manufacturer's instructions, with the thermal cycle program of 95°C for 30 s and 40 cycles of 95°C for 5 s and 60°C for 20 s. Target genes and corresponding primers are listed in Table S1. The levels of the transcripts were evaluated by the relative standard curve method using an α-tubulin gene as internal standard.
Dynamics of inorganic cations and amino acids during polyP accumulation
PolyP rapidly increased from 0.1 to 18.9 μmolc mg−1 protein 4 h after Pi application (Fig. 2a). During polyP accumulation, levels of Na+ and K+ remained constant until 2 h after Pi application, and then increased to 9.5 and 5.7 μmolc mg−1 protein, respectively, 4 h after Pi application (Fig. 2b). The levels of Ca2+ and Mg2+ also remained constant until 2 h after Pi application, and increased to 7.4 and 3.4 μmolc mg−1 protein, respectively, 4 h after Pi application (Fig. 2c). The levels of Fe2+ were an order of magnitude lower than those of the four major cations, and no significant change in the level was observed during polyP accumulation (Fig. S3). The concentrations of Zn2+, Al3+, Cu2+, Ni2+ and Mn2+ were two to several orders of magnitude lower than those of the four major cations, and no consistent results were obtained (data not shown). Arginine was the most abundant free amino acid, followed by asparagine, glutamic acid and alanine (Table S2), which accounted for 53–61, 13–15, 3–5 and 3–4% of the total amino acids, respectively. No significant change in the arginine level was observed during polyP accumulation (Fig. 2d). Asparagine levels were also constant during polyP accumulation, whereas glutamic acid and alanine levels were significantly decreased 1 and 3 h after Pi application, respectively. The levels of other amino acids and ammonium (NH4+) were less than one-twentieth of that of arginine, and did not change during polyP accumulation, except for threonine, glutamine and valine which were decreased slightly after Pi application. During polyP accumulation, cellular ATP levels were constant, and no significant perturbations were observed (data not shown).
The dynamics of the four major cations during polyP accumulation were further investigated from 0 to 24 h after Pi application. PolyP increased from 1.5 to 14.3 μmolc mg−1 protein 4 h after Pi application, and then decreased to the initial level 24 h after Pi application (Fig. 3a). Na+ and K+ increased from 1.1 to 4.2 μmolc mg−1 protein and from 0.4 to 1.8 μmolc mg−1 protein, respectively, 8 h after Pi application, and then decreased to the initial levels 16 h after Pi application (Fig. 3b). Ca2+ and Mg2+ levels increased from 2.8 to 5.7 μmolc mg−1 protein and from 1.8 to 4.3 μmolc mg−1 protein, respectively, 8 h after Pi application, and then decreased to the initial levels 24 h after Pi application (Fig. 3c). The net changes in the total levels of the four cations were compared with the polyP level changes from time zero to 24 h after Pi application based on the data shown in Fig. 3: the total levels of the cations increased and decreased in a near-synchronous and near-equivalent manner to the levels of polyP, although the total levels of the cations reached a maximum c. 4 h later than those of polyP (Fig. 4). The net increase in the level (μmolc mg−1) from 0 to 8 h after Pi application was largest in Na+ (3.1), followed by Ca2+ (2.9), Mg2+ (2.5) and K+ (1.4).
The high-throughput sequencing produced a total of 183 million 101-bp paired-end reads (Table S3). After filtering, 143 million high-quality reads were obtained and assembled into 319 615 contigs to which 57.3% of the filtered reads were uniquely mapped back to the contigs, that is, 43.1% of the reads were mapped to multiple locations in a contig or multiple contigs (Fig. S4; Table S4). Among them, 47 366 contigs showed higher similarities to the predicted gene models and ESTs of R. irregularis than to those of other organisms in the GenBank nucleotide collection, suggesting that they are likely to be of Rhizophagus sp. HR1 origin. To these contigs, 43.1% of the filtered reads (i.e. 75.2% of the uniquely mapped reads) were mapped. In contrast, only 14.2% of the filtered reads were mapped to the rest of the contigs (272 249 contigs), which included not only misassembled contigs and contamination from other organisms, but also transcripts of Rhizophagus sp. HR1 genes that are not conserved between R. irregularis. From the 47 366 putative Rhizophagus sp. HR1 transcripts, a total of 19 144 ORFs (i.e. protein-coding genes) were predicted, to which 31.6 million reads (22.1% of the reads) were uniquely mapped, and thus were subjected to subsequent expression analysis. With reference to the S. cerevisiae and N. crassa databases, 7876 of the 19 144 predicted genes (41.1%) could be functionally annotated. Although the possibility of the presence of splice variants/polymorphisms in the predicted gene set cannot be excluded absolutely, the number of predicted genes in our RNA-Seq analysis is comparable with the number of expressed genes (16 000–18 000) in Rhizophagus spp. (Tisserant et al., 2013), suggesting that variants/polymorphisms are minimal within the predicted genes, and also that the gene set provides a reasonable coverage of the transcriptome in the extraradical mycelia of Rhizophagus sp. HR1. The sequences of putative Rhizophagus sp. HR1 transcripts and predicted genes (ORFs) are available at http://www.agr.hokudai.ac.jp/botagr/rhizo/RhizoCont/Download.html.
The purity of the fungal material obtained from the open pot culture was evaluated on the basis of the proportions of reads mapped to the ribosomal RNA genes (rDNA) of other microorganisms that were contaminated in the fractions. Although c. 45.0 million filtered reads were mapped to contigs similar to the rDNA of various organisms, 94.4% of these reads (42.4 million reads) were mapped to seven contigs (including variants in the divergent domains) that showed the highest similarity to the rDNAs of Rhizophagus sp. HR1 and its close relative (Fig. S5; Table S5). These observations suggested that contamination of transcripts from other organisms was minimal in the RNA-Seq analysis.
Dynamics of gene expression during polyP accumulation
Among the 19 144 predicted genes, significant changes in expression level during polyP accumulation were observed in 2332 genes: 1582 and 750 were up- and down-regulated, respectively, 4 h after Pi application (Fig. S6; Table S6). To evaluate the validity of these results, quantitative RT-PCR was conducted using the same RNA extracts on nine genes responsible for Pi uptake, polyP biosynthesis and cation transport (listed in Table S1). The differential expression levels (fold change in response to Pi application) of the genes observed in the RNA-Seq analysis were significantly correlated with those determined by quantitative RT-PCR (r =0.906, P <0.001), validating the results of RNA-Seq analysis (Fig. S7).
Up-regulated genes were over-represented in 155 GO terms involved in Pi transport, cation transport, polyP metabolism, arginine metabolism, cellular pH regulation and ribonucleotide metabolism (Table S7). Down-regulated genes were overrepresented in 18 GO terms involved mainly in antioxidant reactions and nitrate assimilation/metabolism (Table S8). Genes assigned to the GO terms involved in ATP/energy generation pathways, for example, glycolysis (GO:0006096), pentose phosphate shunt (GO:0006098), tricarboxylic acid (TCA) cycle (GO:0006099), β-oxidation (GO:0006635) and oxidative phosphorylation (GO:0006119), were generally unresponsive to Pi application, or slightly down-regulated (data not shown), except that AAC, which encodes the ADP/ATP carrier of the mitochondrial inner membrane (ORF ID Rh118606 in Table S6), was up-regulated.
For further analysis, detailed profiling of the genes in these GO terms, in addition to those that showed significant changes in expression level, was conducted, with particular emphasis on the dynamics of cations and amino acids during polyP accumulation.
Phosphate uptake and polyP metabolism
Pi application significantly increased the expression of the genes responsible for Pi uptake across the plasma membrane: four of six H+/Pi symporter genes (PHO84) and two Na+/Pi symporter genes (PHO89) (Fig. 5; Table S9). Two of four plasma membrane (P)-type H+-ATPase genes and all four Na+-ATPase genes, which are responsible for driving the H+/Pi and Na+/Pi symporters, respectively, were also up-regulated by Pi application. The expression levels of H+/Na+ antiporter genes (NHA), responsible for Na+ extrusion across the plasma membrane, were also increased by Pi application. The expression of VTC1 and VTC4, responsible for polyP biosynthesis, dramatically increased on Pi application, and, in addition, an endopolyphosphatase gene (PPN) and a vacuolar membrane (V)-type Pi exporter gene (PHO91) were also up-regulated by Pi application.
Various genes responsible for inorganic cation uptake across the plasma membrane were up-regulated by Pi application: K+ transporter genes (KTR), Mg2+ transporter genes (ALR), a Fe3+ permease gene (FTR), a Zn2+ transporter gene (ZRT) and a divalent metal ion transporter genes (SMF). In contrast, no significant changes in the expression levels of Ca2+ channel (CCH) and Ni2+ transporter (NTR) genes were observed. The genes responsible for inorganic cation transport across the vacuolar membrane, a H+/monovalent cation antiporter gene (VNX), H+/Ca2+ antiporter genes (VCX), a Fe2+/Mn2+ transporter gene (CCC), a Zn2+ transporter gene (ZRC), a Zn2+ exporter gene (ZRE) and a Ca2+-ATPase gene were up-regulated in response to Pi application. The expression of several genes encoding the subunits of V-type H+-ATPase, which drives ion transport across the vacuolar membrane, were also increased by Pi application. No significant change in the expression level of the V-type Cu2+ transporter gene (CTR) was observed.
Nitrogen uptake and arginine metabolism
Pi application generally reduced the expression of the genes involved in nitrate assimilation: two NO3− transporter genes (NRT), one of nine NO3− reductase genes (NAR) and two of five NO2− reductase genes (NIR) (Fig. 6; Table S10). By contrast, Pi application generally up-regulated the genes involved in NH4+ uptake, nitrogen assimilation and arginine biosynthesis (urea cycle): one of four NH4+ permease genes (MEP), whose transcripts were most abundant among the four, two glutamine synthetase genes (GS), one of two glutamate synthase genes (GOGAT), a carbamoyl phosphate synthetase gene (CPS), an acetylglutamate kinase and N-acetyl-γ-glutamyl-phosphate reductase (AGK/AGPR), an acetylornithine aminotransferase gene (AOAT), an ornithine carbamoyltransferase gene (OCT), an argininosuccinate synthetase gene (ASS) and an argininosuccinate lyase gene (ASL). Only one of four MEP genes and an ornithine acetyltransferase gene (OAT) were down-regulated after Pi application. The expression of an arginase gene (ARG) and an ornithine transaminase gene (OTA), responsible for arginine degradation, was also increased by Pi application. Interestingly, both a V-type basic amino acid transporter gene (VBA), involved in sequestration into the vacuoles, and two V-type basic amino acid exporter genes (RTC), involved in release from the vacuoles, were up-regulated by Pi application.
The present study demonstrates, for the first time, a global picture of transport and metabolic processes during polyP accumulation in an AM fungus at both the physiological and molecular levels. The application of 1 mM Pi to Pi-starved hyphae induced polyP overplus in the fungus. Although such high concentrations of Pi may rarely occur in natural soils, this approach enhanced the cellular responses to increased Pi availability and thus enabled us to clarify the orchestrated cellular processes involved in Pi acquisition in the symbiotic phase of the fungus. The micro-scale element/amino acid analyses, in conjunction with the transcriptome analysis, revealed that polyP overplus was concurrent with the rapid uptake of inorganic cations, but not with drastic changes in cationic amino acid levels, during which not only the genes responsible for Pi uptake and polyP biosynthesis, but also those responsible for inorganic cation transport, were up-regulated. These results suggest that inorganic cations play a significant role in maintaining cellular homeostasis during polyP accumulation in the fungus.
Transport and metabolic processes responsible for polyP accumulation
The comparative transcriptome analysis revealed that Pi application to Pi-starved hyphae induced the expression of the genes encoding Pi symporters, P-type ATPases and polyP polymerase (VTC complex). Interestingly, PPN, the vacuolar endopolyphosphatase responsible for polyP hydrolysis (Kumble & Kornberg, 1996), and PHO91, a V-type Pi exporter that releases Pi from the vacuoles (Hürlimann et al., 2007), were also up-regulated, supporting the idea that polyP is dynamically turning over during the accumulation and translocation (Ezawa et al., 2001). Although we initially expected that genes involved in the ATP/energy generation pathways would also be up-regulated to supply ATP to polyP biosynthesis, most of the genes involved in this pathway were unresponsive to Pi application. However, the increased expression level of AAC, a mitochondrial ADP/ATP carrier gene that exchanges cytosolic ADP for mitochondrial ATP (Lawson & Douglas, 1988), suggests that ATP export from the mitochondria to the cytosol is increased during polyP accumulation. Given that no perturbation in cellular ATP level was observed during polyP accumulation, the ATP/energy generation pathways are likely to be fully capable of covering the increased ATP demand for polyP overplus without up-regulation of the genes.
The comparative transcriptome analysis also unveiled a complex regulatory mechanism underlying Pi uptake through the two types of high-affinity Pi transporter: H+- and Na+-dependent symporters. In contrast with H+-dependent Pi uptake, less attention has been paid to Na+-dependent Pi uptake in AM fungi (Ezawa et al., 2002). However, the pathway is common in mammals (Collins et al., 2004), protozoa (Dick et al., 2012) and fungi (Versaw & Metzenberg, 1995; Zvyagilskaya & Persson, 2003). In AM fungi, the expression of the gene has only recently been described (Tisserant et al., 2012). A fungal Na+/Pi symporter was first identified as PHO4 in N. crassa (Versaw & Metzenberg, 1995), and then the yeast homologue PHO89 was characterized in detail (Martinez & Persson, 1998; Zvyagilskaya et al., 2008). PHO89 encodes a high-affinity Na+/Pi symporter responsible for Na+-coupled Pi transport with an alkaline pH optimum, which is driven by the electrochemical gradient of Na+ across the plasma membrane generated by Na+-ATPase (Zvyagilskaya et al., 2008). The expression of PHO89 was significantly increased in response to Pi application in Rhizophagus sp. HR1, which was concurrent with the up-regulation of the Na+-ATPase genes, indicating the significance of the Na+-dependent Pi uptake pathway in the Pi acquisition strategy of AM fungi. For the parallel driving of H+- and Na+-dependent pathways, it is likely that at least two types of H+/Na+ antiporter, in addition to the P- and V-type ATPases, are required; one is the P-type antiporter encoded by NHA, and the other is the V-type antiporter encoded by VNX, whose expression was up-regulated in response to Pi application. The former antiporter NHA extrudes Na+ in exchange for H+ across the plasma membrane (Bañuelos et al., 1998), whereas the latter antiporter VNX sequesters Na+ into the vacuoles in exchange for H+ (Cagnac et al., 2007). The coordinated expression of these genes may be responsible for the maintenance of pH/Na+ homeostasis during polyP accumulation.
A moderate concentration of Pi (i.e. 320 μM) up-regulated the H+/Pi symporter gene in Pi-starved extraradical mycelia of R. irregularis in vitro culture, although high concentrations of Pi (> 3 mM) down-regulated the expression, at least within 24 h (Maldonado-Mendoza et al., 2001; Fiorilli et al., 2013). The detachment of mycelia from the roots, however, suppressed the expression, even in the presence of 320 μM Pi, suggesting that the expression is regulated by the sink activity of the host rather than by external Pi (Fiorilli et al., 2013). Given that the host would receive Pi from the fungus at least 8–10 h after Pi application in our culture system (Hijikata et al., 2010), the sink activity was likely to be maintained 0–4 h after Pi application during which the transcript levels were assessed, and thus the expression of the symporter genes was maintained, even in the presence of a high concentration (1 mM) of Pi.
Roles of inorganic cations in polyP accumulation
The time course of the quantitative analysis of cationic components revealed that Na+, K+, Ca2+ and Mg2+ were taken up in a near-synchronous and near-equivalent manner to polyP, providing strong evidence that they play a major role in the neutralization of the negative charge of polyP in the fungal cell. Although the subcellular localization of the increased cations has yet to be elucidated, it is highly likely that large amounts of these cations are sequestered into the vacuoles in which polyP is accumulated for the following reasons: the co-localization of these four cations with P in the vacuoles of R. irregularis has been observed previously (Bücking & Shachar-Hill, 2005); and the genes encoding the V-type inorganic cation transporters are up-regulated in response to Pi application.
The concentrations of polyP, K+ and Ca2+ on the basis of protein (in Fig. 4) can be converted to those on the basis of DW for direct comparison with the data obtained previously (μmolc g−1 DW): polyP, 1230–1700; K+, 370–512; Ca2+, 480–666. Olsson et al. (2008) employed particle-induced X-ray emission analysis in conjunction with ion scanning transmission microscopy for the estimation of element contents in R. irregularis spores grown in vitro: P, 8000; K, 2800; Ca, 2300 μg g−1 DW. These correspond to 258, 72 and 116 μmolc g−1 DW, respectively. The absolute values are largely different between the two studies, probably because they were obtained in different tissue (spores and hyphae) grown under different conditions (in vitro and open pot culture). Nevertheless, the P : K : Ca molar ratios are quite similar between the two studies: 10 : 2.8 : 4.5 in Olsson et al. (2008) and 10 : 3.0 : 3.9 in the present study. These observations suggest that the fungi selectively take up K+ and Ca2+ (and probably also Na+ and Mg2+) to maintain charge neutrality in the cell, independent of the availability of these cations in the environment (culture medium). This idea is indirectly supported by the following results: the net increase in K+ during polyP accumulation was smallest among the four cations, even though Pi was applied in the form of potassium dihydrogen salt; and both Fe2+ and Na+ were applied at 50 μM in the nutrient solution, but the levels of Fe2+ were unresponsive to polyP and less than an order of magnitude lower than those of Na+, suggesting highly selective cation uptake in the fungi. Further experiments under various cation availability conditions are necessary to confirm this idea.
The rapid decrease in the polyP level from 4 h after Pi application suggests that polyP is highly mobile in the cell and thus readily translocated through hyphae towards the plant, as demonstrated previously (Hijikata et al., 2010). To maintain the mobility, the maintenance of the solubility of polyP is essential, which is likely to be regulated by counter ions and pH in the vacuoles. MacDonald & Mazurek (1987) postulated that polyP has two dissociation constants, Ka1 and Ka2, and estimated the values by NMR based on the chemical shifts of the protonated Pi residue; in the presence of a monovalent cation, the pKa1 and pKa2 values of polyP16 (polyP with 16 Pi residues) were 5.0–5.7 and 6.2–6.5, respectively, whereas the addition of a divalent cation to the solution significantly decreased the values to < 4.0 and < 4.5, respectively. Given that the vacuolar pH of R. irregularis might be 5.5–6.0 and constant even after Pi application (Viereck et al., 2004), the presence of a divalent cation in the vacuoles is likely to lead to dissociation of polyP, which would consequently maintain the solubility of polyP.
At the initial phase of polyP accumulation (i.e. from 0 to 4 h after Pi application), however, the total cation levels were c. 30% lower than those of polyP in terms of charge, implying that Pi uptake (and subsequent polyP synthesis) and cation uptake were transiently imbalanced in this phase. During this phase, it seems likely that some of the Pi residues of polyP were temporarily protonated, which was probably assisted through H+ transport by V-type H+-ATPases. Although the solubility of partially (i.e. 30% of Pi residues) protonated polyP has not been investigated experimentally, the fact that total P levels fluctuated in parallel with soluble (urea-extractable) polyP levels after Pi application (Hijikata et al., 2010) suggests that the precipitation of polyP, that is the conversion of ‘soluble polyP’ to ‘insoluble polyP’, was unlikely to occur in this phase.
The synchronous decrease in the four major cations with polyP from 4 h after Pi application suggests that these cations were translocated in association with polyP towards the plant, at least to intraradical hyphae, although our experimental setup did not allow us to address this issue. There have been many reports demonstrating that AM formation increases the concentration of various cations in the host, for example, Na+ (Tian et al., 2004), K+ (Huang et al., 1985), Mg2+ (Giri & Mukerji, 2004) and Cu2+ (Li et al., 1991), which indirectly suggests that metal cations are transferred from the fungi to the host. A direct answer to the question of whether cation transfer between the symbionts occurs could be obtained using radioactive tracers (e.g. 65Zn in Bürkert & Robson, 1994).
Alteration of gene expression in nitrogen uptake and assimilation pathways during polyP accumulation
The role of arginine in neutralizing the negative charge of polyP is limited, at least during polyP overplus. Although arginine levels observed in the present study (100–140 nmol mg−1 DW, converted on the basis of DW) were comparable with those observed in R. irregularis in vitro culture (50–170 nmol mg−1 DW) (Jin et al., 2005), the levels were approximately an order of magnitude lower than those of polyP in terms of electric charge. Pi application, however, altered gene expression in the pathways for nitrogen assimilation and arginine biosynthesis/breakdown. The genes responsible for NO3− uptake (NRT) and subsequent reduction (NAR and NIR) were down-regulated in response to Pi application, and, instead, the NH4+ permease gene MEP was up-regulated. This shift in nitrogen source from NO3− to NH4+ may contribute to the alleviation of the accumulation of negative charge in the cell during massive accumulation of Pi. The up-regulation of the genes responsible for arginine biosynthesis/breakdown pathways in addition to V-type basic amino acid transport/export (VBA/RTC) suggests that de novo synthesis of arginine occurred during polyP accumulation, although no fluctuation in arginine level was detected. One interpretation is that newly synthesized arginine was translocated towards the plants (Govindarajulu et al., 2005), maintaining the cellular arginine levels. Although the accumulation of basic amino acid in the vacuoles is likely to be metabolically independent from that of polyP in fungi (Dürr et al., 1979; Cramer et al., 1980), polyP accumulation may increase the buffering capacity of the vacuoles for cationic compounds, such as arginine, which would consequently activate the biosynthesis and translocation of arginine.
The present study demonstrates the cellular responses of an AM fungus to increased Pi availability, which is the initial process of nutrient delivery in these associations. PolyP overplus, that is the rapid and massive accumulation of polyP in Pi-starved mycelia, was accompanied by near-synchronous and near-equivalent uptake of Na+, K+, Ca2+ and Mg2+, and these cellular events were achieved by the orchestrated regulation of gene expression responsible for mineral uptake, Pi and nitrogen metabolism, and maintenance of cellular homeostasis. Given that the host plant supplies more carbohydrate to a more efficient fungal partner in terms of Pi delivery, namely the ‘fair trade’ (Kiers et al., 2011), those that are capable of delivering larger amounts of Pi are likely to be more competitive, at least under P-deficient conditions. In this context, the capabilities of Pi acquisition and translocation may be directly relevant to competitiveness of individual fungi. Therefore, our study has important implications not only in the physiology, but also in the ecology, of the fungi, and provides a new direction for understanding the mechanism underlying the competitiveness of the fungi.
The PPK-overexpressing E. coli was a kind gift from Dr A. Kornberg (Stanford University, CA, USA). Lotus japonicus MG-20 seeds were supplied through the Frontier Science Research Center of the University of Miyazaki, funded by the National BioResource Project. This work was mainly conducted in the open research facility at the National Agriculture and Food Research Organization/Hokkaido Agricultural Research Center (NARO/HARC) (Y.K., N.H., K.Y., R.O. and T.E.) and supported by a Grant-in-Aid for Scientific Research (22380042) from the Japan Society for the Promotion of Science (T.E.), Grant in Aid for Scientific Research on Innovative Areas (no. 22128001 and 22128006) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (M.K.) and the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (K.S.). We also thank the Functional Genomics Facility for Illumina sequencing and the Data Integration and Analysis Facility for computational resources at the National Institute for Basic Biology.