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Keywords:

  • gene expression;
  • Gigaspora margarita;
  • Glomus intraradices;
  • nutrient excahnge;
  • PHO8

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    We identified a partial cDNA clone showing similarity to the yeast alkaline phosphatase (ALP) gene PHO8 in an EST library constructed from extraradical hyphae of Glomus intraradices.
  • • 
    Using this clone and 5′-RACE (rapid amplification of cDNA ends), we cloned a full-length cDNA for ALP from G. intraradices. By using RT (reverse transcription)–PCR and degenerate primers reflecting conserved regions between the putative amino acid sequences of this cDNA and yeast alkaline phosphatases, we obtained a full-length ALP cDNA from Gigaspora margarita. These genes were designated GiALP and GmALP, respectively.
  • • 
    Both GiALP and GmALP were constitutively expressed in mycorrhizal roots, irrespective of the growth stage, infection rate, and environmental phosphate concentration. The levels of GiALP and GmALP transcripts were higher in mycorrhizal roots than in germinating spores and extraradical hyphae.
  • • 
    These results suggest that arbuscular mycorrhizal ALP is expressed under symbiotic conditions and that it may have a role in nutrient exchange with host plants rather than in nutrient uptake from the rhizosphere.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Arbuscular mycorrhizal (AM) fungi are obligate symbiotic microorganisms that form associations with plant roots in a host nonspecific manner. Most terrestrial plant species live in symbiosis with AM fungi, and AM fungi take up inorganic phosphate (Pi) from soils through extraradical hyphae and transfer the Pi to the host plants. In return, AM fungi receive carbon compounds derived from photosynthesis in host plants (Smith & Gianinazzi-Pearson, 1988; Smith & Read, 1997; Harrison, 1999). This function of AM symbiosis has garnered attention from the viewpoint of sustainable agriculture (Bethlenfalvay & Linderman, 1992). It has been thought that the nutrient exchange between AM fungi and host plants occurs at the arbuscules, which are arbuscular fine hyphae and symbiosis-specific organs.

AM symbiosis is characterized by a specific organ, the arbuscule, which is formed in root cortex cells by the penetration of the finely branched hyphae of AM fungi (Bonfante-Fasolo et al., 1986). This organ is speculated to be a site of nutrient exchange between the host plant and AM fungi (Cox et al., 1980). Phosphate efflux from the fungi to the host plant at arbuscules is supported by the recent discovery of novel plant Pi transporters that are localized around arbuscules and acquire Pi from the fungi (Rausch et al., 2001; Harrison et al., 2002; Paszkowski et al., 2002).

The alkaline phosphatase (ALP) of AM symbiosis has been investigated since a mycorrhizal-specific ALP was identified electrophoretically in a crude extract of mycorrhizal roots (Gianinazzi-Pearson & Gianinazzi, 1976, 1978). Histochemical evidence of strong ALP activities at arbuscules (Gianinazzi et al., 1979; Tisserant et al., 1993; Ezawa et al., 1995) has raised the idea that the ALP is involved in phosphate efflux from arbuscules. ALP activity was not observed in host plants and may originate in AM fungi in mycorrhizal roots (Gianinazzi-Pearson & Gianinazzi, 1978). This hypothesis was later confirmed through a combined electrophoretic–enzymatic hyphal separation technique (Kojima et al., 1998). Therefore, ALP activity has been used as a marker for the metabolic activity of AM fungi (Tisserant et al., 1993; Guillemin et al., 1995).

Recently it was found that the efflux of phosphate from intraradical hyphae separated from roots by enzymatic digestion was partly suppressed by inhibitors of ALP (T. Kojima, unpublished data), suggesting that ALP in arbuscules may have an important role in the transfer of phosphate from AM fungi to host plants. Because purification of the enzyme has so far been unsuccessful (Kojima et al., 2001), little is known about the enzymatic characteristics of the ALP in AM fungi (Gianinazzi-Pearson & Gianinazzi, 1976). A specific inhibitor, Be2+, was used to characterize the ALP in intraradical hyphae, and these efforts revealed that this ALP has increased affinity for sugar phosphate and does not hydrolyse polyP (Ezawa et al., 1999). However, the function of the arbuscular ALP in symbiosis is still little known, and cloning of the enzyme may shed light on its unknown function.

Molecular genetic information on AM fungi has been accumulated. The development of high-throughput DNA sequencing techniques and high-quality RT–PCR kits have enabled the construction of expressed sequence-tagged (EST) libraries from relatively small samples of AM fungi, and EST libraries from the mycorrhizal roots, germinating spores, and extraradical hyphae of these organisms have been constructed (Franken & Requena, 2001; Sawaki & Saito, 2001). Such an extensive approach may facilitate or accelerate the identification of as-yet unknown genes specific to mycorrhizal symbiosis. In this regard, a cDNA clone showing similarity to a yeast ALP gene (PHO8) (Kaneko et al., 1987) was found in an EST library constructed from the extraradical hyphae of Glomus intraradices. Using this clone, we succeeded in cloning the ALP genes from the AM fungi G. intraradices and Gigaspora margarita for the first time. Here we present the characteristics of these ALP genes and their expression.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mycorrhizal plants

The growth medium for mycorrhizal plants was a mixture (5 : 4 : 1 by volume) of a humus-rich Ando soil, sand, and a commercial horticulture medium (Kureha Chemical Co., Japan), with 1 g l−1 CaCO3 added. Before the components of the growth medium were mixed, the sand and horticulture medium were autoclaved for 20 min at 120°C, and the soil was autoclaved for 1 h at 121°C.

Onion (Allium cepa L. cv. Sensyuchukouki) seeds were surface-sterilized in 0.5% (w/v) NaOCl solution for 1 h and germinated on moist filter paper. Onion seedlings were transplanted to pots filled with growth medium and inoculated with G. intraradices isolate DAOM1862 or G. margarita isolate MAFF 520054. For G. intraradices, 50 mg of onion mycorrhizal roots per pot was used as the inoculum. For G. margarita, 50 spores per pot were used as the inoculum. The inoculated plants were grown in a growth cabinet with a 14-h day at 23°C and a 10-h night at 20°C. Some mycorrhizal plants were supplemented with 1 mm KH2PO4, daily beginning 5 weeks after inoculation. At harvest, duplicate samples of 5–10 mycorrhizal plants were collected, and mycorrhizal roots and extraradical hyphae were separated in ice-cold water by using forceps; the hyphae were briefly sonicated and then immediately frozen in liquid N2. Subsamples of mycorrhizal roots were used for RNA isolation (see following) and for Trypan blue staining (Phillips & Hayman, 1970) to measure colonization.

Germination of spores

The growth medium was embedded in autoclaved 1% agar in plastic Petri dishes and then covered with cellulose acetate membranes (0.8 µm pore size) (Uetake et al., 2002). Spores of G. intraradices and G. margarita were placed on the membranes. The plates were inverted and incubated in the dark at 23°C for 8 h followed by 25°C for 16 h daily. At 2 weeks after germination, germinating spores with germ tubes were collected and immediately frozen in liquid N2.

Construction of EST library from extraradical hyphae of G. intraradices

The EST library was constructed by The Samuel Robert Noble Foundation (Admore, Oklahoma, USA). Total RNA was extracted from extraradical mycelia of G. intraradices grown in a two-compartment Petri plate system in association with Daucus carota-transformed roots. cDNA was prepared from total RNA using the SMART cDNA synthesis system (Clontech, Franklin Lakes, NJ, USA). The cDNA was directionally ligated into the lambda TriplEX2 vector (Clontech) and packaged using Gigapack Gold packaging extracts (Stratagene, La Jolla, CA, USA). Plasmids containing cDNA inserts were obtained from recombinant lambda TriplEX2 phage via Cre–lox mediated conversion in BM25.8 cells (Clontech).

RNA isolation

The total RNA used in the cloning of ALP cDNAs was isolated from G. intraradices and G. margarita mycorrhizal roots by phenol–SDS extraction and LiCl precipitation (Shirzadegan et al., 1991). Poly(A)+RNA was isolated using Oligotex-dT30 (Takara, Tokyo, Japan). Total RNA used in the expression analysis by real-time RT–PCR was isolated from each tissue by using RNeasy plant mini kits (Qiagen, Hilden, Germany).

RT–PCR and cloning of products

First-strand cDNA was synthesized by reverse transcription of mRNA isolated from mycorrhizal roots by using oligo-dT12-18 primers (Amersham Pharmacia, Piscataway, NJ, USA) and reverse transcriptase (Superscript II, Invitrogen, Carlsbad, CA, USA). Partial cDNA fragments were amplified by PCR by using the first-strand cDNA as a template and the appropriate primers. Amplification was carried out using Taq DNA polymerase (AmpliTaq Gold, Perkin-Elmer, Wellesley, MA, USA). The PCR products were ligated into T-vectors (pGEM-T Easy Vector, Promega, Madison, WI, USA) for subsequent analysis.

5′- and 3′-RACE

To obtain full-length cDNA fragments, the partial cDNA fragments were lengthened by the 5′- and 3′-rapid amplification of cDNA ends (RACE) method by using the SMART RACE cDNA Amplification Kit (Clontech) with appropriate primers and total RNA isolated from mycorrhizal roots, according to the manufacturer's recommendations.

Cloning of cDNA for G. intraradices ALP

We isolated a partial cDNA clone showing similarity to yeast PHO8 from the EST library from extraradical mycelia of G. intraradices. The cDNA lacked the 5′ region, although it had a poly(A) tail at the 3′ end. This partial cDNA was lengthened by 5′-RACE by using a cDNA-specific primer (Table 1) and total RNA isolated from G. intraradices mycorrhizal roots at 6 weeks after inoculation to obtain the full-length cDNA (GiALP).

Table 1.  Primers used in this study
5′-RACE for GiALP5′-CATGTGTCGCCCACCCAAGTTGAG-3′
RT–PCRfor partial cDNA of GmALP
Sense5′-GAAGGVWSYAGRATHGA-3′
Antisense5′-CCACCVGTTTCRTGRTC-3′
5′, 3′-RACE for GmALP
5′-RACE5′-TGACCGATATTACGACTGTTCCCGGATG-3′
3′-RACE5′-AACCGGTAGTTGCAGAAGTGATGATCGG-3′
PCR for GmALP ORF
Sense5′-TTATCTTTGAGTGTGCCATG-3′
Antisense5′-ACCATTCCATGAATCGAG-3′
Real-time PCR
GiALP
Sense5′-GCTCGTCAAGTTTCCGATCTAC-3′
Antisense5′-CTCCGATTCCTAATCCGCTAC-3′
GmALP
Sense5′-ACCGAATATCTTAACTTGGATCCTG-3′
Antisense5′-AAGTTCGGCGAGGTATATGACC-3′
18S rRNA gene of G. intraradices
Sense5′-GGATAACCGTGCTAATTCTAGAGC-3′
Antisensetaxon-specific primer; VAGLO (Simon et al., 1993)
18S rRNA gene of G. margarita
Sense5′-GGATAACCGTGCTAATTCTAGAGC-3′
Antisensetaxon-specific primer VAGIGA (Simon et al., 1993)

Cloning of cDNA for G. margarita ALP

A pair of degenerate primers (Table 1) was designed based on the amino acid sequences of highly conserved regions within the ALPs of G. intraradices, Saccharomyces cerevisiae, and Shizosaccaromyces pombe. An ALP gene-specific fragment was amplified by RT–PCR using these degenerate primers and the mRNA isolated from G. margarita mycorrhizal roots at 6 weeks after inoculation. This cDNA fragment was lengthened with 5′- and 3′-RACE using two cDNA-specific primers (Table 1) and total RNA from G. margarita mycorrhizal roots to obtain the full-length cDNA (GmALP).

Sequence analysis

DNA sequence analysis was performed through dideoxy sequencing in a DNA sequencer (DSQ-2000L, Shimadzu, Tokyo, Japan). Homology searches were carried out using the BLASTX and FASTA programs on the DDBJ server (http://www.ddbj.nig.ac.jp/). Searches for highly conserved regions among amino acid sequences were carried out using the BLOCKS database on the Blocks WWW server (http://blocks.fhcrc.org/blocks). Searches for active sites and N-linked glycosylation sites were carried out using the PROSITE database on the ExPASy Molecular Biology server (http://sexpasy.org/). Hydropathy plots were carried out using the program TMpred (Hofmann & Stoffel, 1993) on the Swiss EMBnet server (http://www.ch.embnet.org/). Phylogenetic analysis was conducted by using the programs ClustalW (Thompson et al., 1994) and TREEVIEW (Page, 1996).

Southern blot analysis

Genomic DNA was isolated from spores of G. margarita by using a slightly modified version of the method of van Buuren et al. (1999). We digested 3 µg of genomic DNA with each of the endonucleases EcoRI, EcoRV, HindIII, and ScaI, fractionated the samples on a 1% agarose gel, and blotted them onto a positively charged nylon membrane (Roche, Mannheim, Germany) according to standard procedures (Sambrook et al., 1989). The membranes were hybridized overnight with digoxigenin-labelled probes in an appropriate hybridization buffer (DIG Easy Hyb, Roche) at 40°C. After hybridization, the blot was washed twice in 2× SSC/0.1% SDS at room temperature for 5 min and twice in 0.5× SSC/0.1% SDS at 65°C for 15 min each wash. Signals on the blot were detected by the chemiluminescent method using DIG Luminescent Detection Kit (Roche) and exposed to X-ray film. The probe for the GmALP open reading frame (ORF) was obtained by PCR amplification using the cDNA clones as templates with specific primers (Table 1).

Real-time PCR quantification of gene expression

The expression of ALP and 18S rRNA genes was monitored by the real-time PCR method using LightCycler (Roche) after reverse transcription of total RNA. Total RNA was isolated from mycorrhizal roots, extraradical mycelia, and germinating spores of G. intraradices and G. margarita. First-strand cDNA was reverse transcribed from 250 ng of total RNA with 10 mm random hexamers (Amersham Pharmacia) in a 20-µl reaction volume containing reverse transcriptase (Omniscript, Qiagen) according to the manufacturer's recommendations. For a negative control, a reaction lacking reverse transcriptase (‘No-RT’ reaction) was also carried out. Real-time PCR was carried out using QuantiTect SYBR Green PCR (Qiagen) with gene-specific primers (Table 1) and aliquots (1 µl each) of the No-RT and RT-containing reactions as template. The plasmid DNA containing each amplicon was used as a standard for quantifying the copy number of each gene. Because small amounts of genomic DNA contaminated the total RNA preps, the copy number for the No-RT reaction was subtracted from that of the RT reaction to determine the copy number derived from total RNA. Expression levels of ALP were evaluated by the ratio of copy number of ALP to that of 18S rRNA.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cloning a full-length cDNA for the G. intraradices ALP

From an EST library from the extraradical mycelia of G. intraradices, we identified an EST clone (Gi08A10) with similarity to the S. cerevisiae repressible ALP encoded by the PHO8 gene (Kaneko et al., 1987). The cDNA insert in the Gi08A10 clone was about 650 bp in length and had a poly(A) tail at the 3′ end. Several amino acid domains are highly conserved between the deduced amino acid sequences of the cDNA insert in Gi08A10 and yeast PHO8, and these two sequences had 32% identity with each other. From these results, we surmised that the insert of Gi08A10 was a partial cDNA that encoded the C-terminal portion of ALP. Therefore, we lengthened this partial cDNA with 5′-RACE using a cDNA-specific primer and total RNA isolated from G. intraradices mycorrhizal roots; this process enabled us to obtain the full-length cDNA for the ALP from G. intraradices (GiALP). GiALP is 1829 bp long and contains an ORF encoding a 525-amino-acid polypeptide (molecular mass, 59.1 kDa). The ORF of GiALP is flanked by 146 bp of untranslated sequence at the 5′ end and by 178 bp of untranslated sequence including the poly(A) tail at the 3′ end. We submitted the nucleotide sequence of GiALP to EMBL/GenBank/DDBJ under the accession number AB114298.

Cloning a full-length cDNA for the G. margarita ALP

A partial cDNA fragment was obtained by RT–PCR with a pair of degenerate primers and mRNA from mycorrhizal roots of G. margarita. This cDNA fragment was 158 bp long and showed 79.4% identity in nucleotide sequence with GiALP. This partial cDNA was lengthened by 3′-RACE using a cDNA-specific primer and total RNA from G. margarita mycorrhizal roots and then a cDNA fragment containing the poly(A) tail. Then the cDNA fragment lacking the 5′ end was lengthened by the 5′-RACE using a second cDNA-specific primer to yield the full-length cDNA (GmALP). GmALP is 2004 bp long and contains an ORF encoding a 539-amino-acid polypeptide (molecular mass, 60.3 kDa). The ORF of GmALP is flanked by 34 bp of untranslated sequence at the 5′ end and by 350 bp of untranslated sequence including the poly(A) tail at the 3′ end. The nucleotide sequence of GiALP has been submitted to EMBL/GenBank/DDBJ under the accession number AB114299.

Structures of GiALP and GmALP

GiALP and GmALP are 68.9% similar in their nucleotide sequences within the coding regions, and the two putative polypeptides are 64.1% identical in their deduced amino acid sequences (Table 2). BLASTX and FASTA searches showed that GiALP and GmALP have the highest degree of similarity with yeast ALPs. GiALP shares 45.9% and 42.3% amino acid identity with the ALPs from S. pombe and S. cerevisiae, respectively. GmALP shares 46.5% and 42.9% amino acid identity with the ALPs from S. pombe and S. cerevisiae, respectively.

Table 2.  Percentage of identity between the deduced amino acid sequences of alkaline phosphatases (ALPs) from arbuscular mycorrhizal fungi and yeasts
 % Identity
GiALPGmALPPHO8S. pombeAccession no.
  1. GiALP, ALP from Glomus intraradices; GmALP, ALP from Gigaspora margarita; PHO8, ALP from Saccharomyces cerevisiae; S. pombe, ALP from Shizosaccaromyces pombe.

GiALP100 64.1 42.3 45.9AB114298
GmALP 100 42.9 46.5AB114299
PHO8  100 44.5M21134
S. pombe   100AF316541

A BLOCKS search revealed that six domains are highly conserved among known ALPs of various organisms, GiALP, and GmALP (Fig. 1). Furthermore, a PROSITE search revealed a domain for the ALP active site and several consensus sites for N-linked glycosylation in both GiALP and GmALP (Fig. 1). Hydropathy plots (data not shown) of the deduced amino acid sequences suggest that like yeast ALPs, GiALP and GmALP each have a membrane-spanning region at the N-terminus (Fig. 1). The phylogenetic tree showed that GiALP, GmALP, and yeast ALPs are in the same cluster (Fig. 2).

image

Figure 1. Deduced amino acid sequences of arbuscular mycorrhizal fungal and yeast alkaline phosphatases (ALPs). Identical amino acids are indicated by asterisks and conserved substitutions are indicated by dots. Boxed sequences are regions highly conserved among the known ALPs. Shaded sequences are consensus active sites for ALP. Sequences underlined with dashed lines are consensus sites for N-linked glycosylation. Sequences underlined with solid lines are membrane-spanning domains, as predicted by TMpred.

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image

Figure 2. Predicted phylogenetic relationship among alkaline phosphatases (ALPs). Phylogenetic tree was constructed on the basis of predicted amino acid sequences of ALPs of arbuscular mycorrhizal fungi, Saccharomyces cerevisiae (EMBL/GenBank/DDBJ accession number, M21134), S. pombe (AF316541), Bacillus licheniformis (U79570), Lactobacillus delbrueckii (AF320303), Escherichia coli (M29669), Pyrococcus abyssi (AJ248286), Halobacterium sp. (AE005160), Drosophila melanogaster (NM170534), silkworm (D90454), mouse (intestinal, M61705; nonspecific, J02980), and human (intestinal, M61705; placental, M12551; placental-like, X55958; nonspecific, M24439).

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Genomic Southern blot analysis with the entire ORF of the GmALP cDNA showed that a single distinct band was detected when the genomic DNA of G. margarita was digested with EcoRV, HindIII, and ScaI and that three distinct bands were detected when the genomic DNA was digested with EcoRI (Fig. 3). There was no recognition site for EcoRV, HindIII, or ScaI in the GmALP ORF, but there were two EcoRI sites. These results reveal that a PHO8-like ALP gene is present as a single-copy gene in the G. margarita genome. We could not evaluate the gene copy number for G. intraradices because we were unable to isolate sufficient genomic DNA.

image

Figure 3. Southern blot analysis of Gigaspora margarita genomic DNA digested with EcoRI, EcoRV, HindIII, and ScaI. The blots were hybridized with a probe comprising the entire open reading frame of GmALP.

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Expression of GiALP and GmALP genes

The expression of the GiALP and GmALP genes was compared among the mycorrhizal roots, extraradical hyphae, and germinating spores through real-time PCR after reverse transcription of total RNA (Fig. 4). The expression of GiALP in mycorrhizal roots was about 6 times higher than that in extraradical hyphae and about 7 times higher than that in germ spores. The expression of GmALP in mycorrhizal roots was about 4 times higher than that in extraradical hyphae and about 3 times higher than that in germ spores. GiALP and GmALP genes were not expressed in nonmycorrhizal roots (data not shown).

image

Figure 4. Expression of alkaline phosphatase (ALP) genes in mycorrhizal roots, extraradical hyphae, and germinating spores. Total RNA was isolated from mycorrhizal roots and extraradical hyphae of mycorrhizal plants at 6 weeks after inoculation and from 2-week-old germinating spores. The amounts of transcripts of ALP and 18S rRNA in the total RNA from each tissue were estimated by real-time RT–PCR. The transcripts of ALP and 18S rRNA were not detected in the total RNA from nonmycorrhizal roots (data not shown). Bar, 1 sd of the mean (n = 3).

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To investigate the relation between the expression levels of the ALP genes and the colonization rates of AM fungi, we determined the expression of GiALP and GmALP in the mycorrhizal roots and extraradical hyphae weekly beginning 2 weeks after inoculation (Fig. 5). Although the colonization rates increased, the transcript levels of GiALP and GmALP were constitutively high in the mycorrhizal roots and constitutively low in the extraradical hyphae.

image

Figure 5. Expression of alkaline phosphatase (ALP) genes in mycorrhizal roots (closed bars) and extraradical hyphae (open bars) at different growth stages. Total RNA was isolated from each tissue at the indicated growth stages. The amounts of transcripts of ALP and 18S rRNA in the total RNA from each tissue were estimated by real-time RT–PCR. In addition, infection rates were measured at harvest. Bar, 1 sd of the mean (n = 3).

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To investigate the relation between the expression levels of the ALP genes and the environmental Pi concentration, mycorrhizal plants grown under low Pi conditions were supplemented with 1 mm Pi daily beginning 6 weeks after inoculation (Fig. 6). The transcript levels of GiALP and GmALP were constitutively high in the mycorrhizal roots, irrespective of the environmental Pi concentration.

image

Figure 6. Effect of adding Pi on the expression of alkaline phosphatase (ALP) genes in mycorrhizal roots. Total RNA was isolated from mycorrhizal plants supplemented with 0 mm (closed bars) or 1 mm (open bars) Pi daily beginning 5 week after inoculation. The amounts of transcripts of ALP and 18S rRNA in the total RNA from each tissue were estimated by real-time RT–PCR. Bar, 1 sd of the mean (n = 3).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We successfully cloned full-length cDNAs for the ALPs of two AM fungi by using an EST library of G. intraradices and a cDNA similar to the yeast ALP PHO8. PHO8-like ALPs occur in a wide range of organisms (with the exception of plants), constituting the ALP family (accession number in the BLOCKS database, IPB001952). The amino acid sequences of the known PHO8-like ALPs included in this family share six conserved domains. These six domains are also well conserved in the AM fungal ALPs that we isolated, GiALP and GmALP (Fig. 1).

The secondary structures of GiALP, GmALP, and yeast ALPs are very similar. In particular, the hydrophobic domain, which is thought to be the transmembrane domain, is present in the N-terminal regions of GiALP, GmALP, and yeast ALPs (Fig. 1). The yeast PHO8 enzyme is localized in vacuoles and attached to tonoplasts (Clark et al., 1982). In this regard, the enzymes encoded by GiALP and GmALP may also be membrane proteins.

The overall homology between the amino acid sequences of GiALP, GmALP, and yeast ALPs is not high (Table 2), because the sequences beyond the six conserved domains are not well conserved (Fig. 1). However, the phylogenetic tree reveals that GiALP, GmALP, and yeast ALPs are in the same cluster (Fig. 2). In light of the similarity in the positions of the conserved domains, similar secondary structures, and phylogenetic relationship, we are convinced that GiALP and GmALP are ALP genes of AM fungi.

It is interesting that there is only one copy of the GmALP gene (Fig. 3). Because spores of AM fungi contain multiple nuclei, it is surprising that we found only one copy of GmALP even though we used the full-length cDNA as a probe.

We tried to complement ALP activity in a PHO8 deletion mutant of S. cerevisiae (Saccharomyces deletion project, http://www-deletion.stanford.edu/) by introducing GiALP or GmALP. Although the gene was expressed in the transformed yeast, ALP activity was not complemented (data not shown). Yeast ALP is a N-glycosylated protein composed of two identical subunits with an Mr of 66 000, of which the carbohydrate component accounts for about 8% (Onishi et al., 1979). Yeast ALP is synthesized as an inactive form. The ALP protein becomes an active form by carbohydrate modification and removal of C-terminal peptide through delivery of ALP protein from ER to vacuole (Klionsky & Emr, 1989). Probably the GiALP and GmALP proteins in yeast cells may not be processed appropriately into an active form.

Are the ALPs encoded by GiALP and GmALP identical with the ALP revealed by the enzymatic–cytohistochemical technique? ALP activity was cytochemically detected in the vacuoles in arbuscules of AM fungi (Gianinazzi et al., 1979), and the ALP activity in the insoluble fraction was higher than that in the soluble fraction when proteins were extracted from AM fungal tissues (Ezawa et al., 1999). These results suggest that like the yeast PHO8 ALP, the cytohistochemically detected ALP in AM fungi may be a membrane protein presumably attached to tonoplast. Biochemical analysis revealed that the ALP crudely extracted from AM fungi was nonspecific as to substrate, although the enzyme had a greatly increased affinity for sugar phosphate (Ezawa et al., 1999). Yeast PHO8 ALP is known to be nonspecific regarding substrate (Kaneko et al., 1985).

The transcript levels of GiALP and GmALP in mycorrhizal roots were much higher than those in extraradical hyphae and germinating spores (Figs 5 and 6). Microscopic observation showed that the intraradical hyphae had a higher proportion of ALP-active hyphae than did the extraradical hyphae (Zhao et al., 1997; Kjøller & Rosendahl, 2000; van Aarle et al., 2002). Enzyme activities in crude extracts showed higher ALP activity in intraradical hyphae than in germinating spores (Saito, 1995). Furthermore, the GiALP and GmALP genes were constitutively expressed irrespective of growth stage and colonization rate (Fig. 5). Tisserant et al. (1993) and Zhao et al. (1997) showed that the proportions of intraradical hyphae of Glomus sp. and Gigaspora sp. having ALP activity approximately correlated with the colonization rates. In fact, the levels of ALP activity in the intraradical hyphae of Glomus sp. and Gigaspora sp. were relatively stable regardless of the growth stage (Boddington & Dodd, 1999). These tendencies in the localization and activity of the ALPs in AM fungi agreed with the observed expression patterns of GiALP and GmALP, suggesting that these genes may encode the ALP observed with the enzymatic–histochemical technique.

The yeast ALP encoded by PHO8 is a repressive type, and its transcription is enhanced by Pi deficiency (Kaneko et al., 1985; Oshima, 1997), indicating that the PHO8 ALP has a role in the hydrolysis of stored phosphorus compounds under conditions of phosphorus deficiency. Because the ALPs in AM fungi might have a similar function, we evaluated the effect of external Pi conditions on the expression of GiALP and GmALP (Fig. 6). However, the expression of both genes was constitutive irrespective of Pi addition.

Olsson et al. (2002) examined the effect of external Pi on the metabolism of G. intraradices in carrot hairy roots and found that the proportion of ALP-active extraradical hyphae was increased with high-Pi medium. Boddington & Dodd (1998, 1999) used a conventional soil pot culture system and reported that the ALP activity in the intraradical hyphae of Gl. manihotis grown under Pi-sufficient conditions was lower than that under Pi-deficient conditions, although the ALP activity in the intraradical hyphae of Gi. rosea was not influenced by the Pi levels. van Aarle et al. (2002) found that Pi addition to established AM-inoculated onion did not influence the proportion of ALP in either the intraradical or extraradical hyphae of G. margarita. The apparent inconsistency among these experiments may be due to differences between the experimental system or the plant–fungus combination. Because we followed the same experimental system as van Aarle et al. (2002), it is plausible that our results (Fig. 6) agree with theirs.

In the yeast S. cerevisiae, the expression of several genes associated with the PHO regulatory system, which responds to changes in the Pi concentration, are controlled at the transcriptional level. The yeast PHO regulatory system includes the expression of genes encoding structural proteins such as ALP (PHO8), acid phosphatase, and a high-affinity Pi transporter, and positive and negative regulatory factors of various structural proteins (Lemire et al., 1985; Toh-e et al., 1988; Bun-ya et al., 1991; Lenburg & Oshea, 1996; Oshima et al., 1996). Maldonado-Mendoza et al. (2001) showed that, as in yeast, the expression of a Pi transporter gene in the extraradical hyphae of G. intraradices was regulated in response to the environmental Pi concentration. This finding suggests that AM fungi may have a regulatory system similar to the yeast PHO system, implying that the expression of the ALP genes of AM fungi may be regulated in response to the environmental Pi concentration. However, the expression of GiALP and GmALP was not affected by the environmental Pi concentration. Unlike yeast cells, AM fungal cells are differentiated, and the environments of the intraradical and extraradical hyphae are very different. The ALP genes we identified were mainly expressed in mycorrhizal roots (i.e. intraradical hyphae). However, the Pi transporter gene was expressed in extraradical hyphae (Maldonado-Mendoza et al., 2001). These results suggest the regulatory system in AM fungi may be more complex than the yeast PHO regulatory system.

The present study revealed that the ALP genes in AM fungi (especially in the intraradical hyphae) are constitutively expressed under symbiotic conditions, irrespective of colonization rates and environmental Pi concentration. These results suggest that ALP may have a significant function specific to the intraradical hyphae.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported in part by Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of Bio-oriented Technology Research Advancement Institution (BRAIN) of Japan.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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