Journal of Experimental Botany, Vol. 51, No. 348, pp. 1179-1188,
July 2000
© 2000 Oxford University Press
Original papers |
Induced activity of adenine phosphoribosyltransferase (APRT) in iron-deficient barley roots: a possible role for phytosiderophore production
1 Laboratory of Plant Molecular Physiology, Department of Applied Biological Chemistry, The University of Tokyo, 1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan
2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 2–1-6 Sengen, Tsukuba 305–0047, Japan
Received 15 October 1999; Accepted 2 February 2000
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Abstract |
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To isolate the genes involved in the response of graminaceous plants to Fe-deficient stress, a protein induced by Fe-deficiency treatment was isolated from barley (Hordeum vulgare L.) roots. Based on the partial amino acid sequence of this protein, a cDNA (HvAPT1) encoding adenine phosphoribosyltransferase (APRT: EC 2.4.2.7) was cloned from a cDNA library prepared from Fe-deficient barley roots. Southern analysis suggested that there were at least two genes encoding APRT in barley. Fe deficiency increased HvAPT1 expression in barley roots and resupplying Fe to the Fe-deficient plants rapidly negated the increase in HvAPT1 mRNA. Analysis of localization of HvAPT1- sGFP fusion proteins in tobacco BY-2 cells indicated that the protein from HvAPT1 was localized in the cytoplasm of cells. Consistent with the results of Northern analysis, the enzymatic activity of APRT in barley roots was remarkably increased by Fe deficiency. This induction of APRT activity by Fe deficiency was also observed in roots of other graminaceous plants such as rye, maize, and rice. In contrast, the induction was not observed to occur in the roots of a non-graminaceous plant, tobacco. Graminaceous plants generally synthesize the mugineic acid family phytosiderophores (MAs) in roots under Fe-deficient conditions. In this paper, a possible role of HvAPT1 in the biosynthesis of MAs related to adenine salvage in the methionine cycle is discussed.
Key words: APRT, barley, Fe deficiency, methionine cycle, mugineic acid.
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Introduction |
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Mugineic acid family phytosiderophores (MAs) are natural chelators of Fe(III) and produced specifically by graminaceous plants (Takagi et al., 1984
). Although Fe(III) in soils forms sparingly soluble Fe such as Fe(OH)3, MAs can solubilize these inorganic Fe(III)-compounds by chelation. Graminaceous plants secrete MAs from roots into the rhizosphere and the MAs chelating Fe(III) are reabsorbed by their roots. This direct mechanism for acquiring Fe(III) with MAs is classified as a Strategy-II mechanism, while the mechanism of non-graminaceous plants is called Strategy-I (Marschner et al., 1986). The biosynthetic pathways of the MAs group have been determined (Shojima et al., 1990). Methionine (Met) is the primary precursor of MAs (Mori and Nishizawa, 1987).
In order to isolate genes encoding enzymes involved in the biosynthesis of MAs, three different approaches using barley roots have been chosen: protein purification of the key enzymes, differential screening of genes between Fe-deficient and Fe-sufficient barley roots, and differential screening of proteins by two-dimensional (2D)-PAGE analysis of the proteins in Fe-deficient and Fe-sufficient barley roots. Protein purification was successful for two of the enzymes crucial to MAs biosynthesis. Nicotianamine synthase (NAS) (Higuc et al.,1999) and nicotianamine aminotransferase (NAAT) (Takahashi et al., 1999) were purified from Fe-deficient barley roots and the corresponding genes were isolated from a cDNA library prepared from Fe-deficient barley roots. Differential screening identified four genes, Ids1, Ids2, Ids3 (iron deficiency specific genes), and IDI1 (GenBank accession nos. X58540, D10057, AB024058 and AB025597, respectively), whose expression was induced by Fe deficiency. Among them, Ids2 and Ids3 are thought to participate in the hydroxylation of deoxymugineic acid (DMA) in the biosynthesis of other MAs (Okumura et al., 1994; Nakanishi et al., 1993, 1997). Ids3 is a putative gene encoding the enzyme that catalyses the conversion of DMA to mugineic acid (MA). IDI1 is presumed to be a gene involved in the Met cycle, which generates Met, the precursor needed for the biosynthesis of MAs (Yamaguchi et al., 2000).
As reported previously (Suzuki et al., 1998), comparison of 2D-PAGE gels from Fe-sufficient and Fe-deficient barley roots demonstrated that the presence of several proteins increased under Fe-deficient conditions. Some of these were collected from gels and their amino acid sequences were partially determined. Protein ‘W’ was revealed to be formate dehydrogenase and protein ‘Y’ was found to be encoded by Ids3 (Suzuki et al., 1998). The function of protein ‘D’ (a 36 kDa peptide) remains unknown (Suzuki et al., 1997). Protein ‘C’ was assumed to be an APRT protein based on its partial amino acid sequences (Suzuki et al., 1998). In this paper, the cloning of a cDNA encoding protein ‘C’ in order to examine its relationship to the biosynthesis of MAs is reported. The idea is discussed that the expression of this gene, named HvAPT1, has an important role related to the Met cycle, which supplies the Met needed for MAs production.
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Materials and methods |
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Plant material
All plants were grown hydroponically by the method of Suzuki et al. (Suzuki et al., 1998
). Barley (Hordeum vulgare L. cv. Ehimehadaka no. 1) and rye (Secale cereale L.) were grown in a growth chamber at 19 °C under artificial light. Rice (Oryza sativa L. cv. Nipponbare) and maize (Zea mays L. cv. Alice) were grown in a greenhouse at 30 °C under natural light. Tobacco (Nicotiana tabacum L. var. SR1) were grown in a greenhouse at 25 °C under natural light. After 2–3 weeks of growth with Fe(III)-EDTA (10 µM), plants were grown without Fe for 2–3 weeks, until the leaves appeared severely chlorotic (a symptom of Fe deficiency). The leaves and roots were then harvested and stored at -80 °C until use. 10 µM Fe(III)-EDTA was used to resupply Fe to the Fe-deficient barley plants.
Protein digestion and amino acid sequence analysis
The comparison of 2D-PAGE gels from Fe-sufficient and Fe-deficient barley roots demonstrated the existence of protein ‘C’, which increased under Fe-deficient conditions. All the procedures used for protein ‘C’ analysis, such as collection from 2D-PAGE gels, digestion by cyanogen bromide and lysylendopeptidase, and amino acid sequence analysis, were the same as reported previously (Suzuki et al., 1998).
Cloning the cDNA encoding APRT
For the PCR template, cDNAs made from poly(A)+ RNA isolated from Fe-deficient barley roots was used. These cDNAs were produced by reverse transcription using an antisense primer called the (dT)17-adapter primer (Frohman et al., 1988). A degenerate primer based on the partial amino acid sequence was designed as a sense primer, 5'-AARGGIAARCCIGGIGARGTIAT. PCR was performed using the adapter primer and the degenerate sense primer. A 550 bp long PCR product with the deduced amino acid sequence of protein ‘C’ was identified.
A cDNA library prepared from poly(A)+ RNA isolated from Fe-deficient barley roots was screened by colony hybridization. The 550 bp PCR fragment was labelled with [
-32P]dATP using a random primer labelling kit (Ver. 2, TaKaRa, Tokyo). Nylon membranes (Hybond-N, Amersham) were hybridized at 65 °C overnight. The basic hybridization buffer contained 250 mM NaH2PO4-H3PO4 (pH 7.2), 7% (w/v) SDS, and 1 mM EDTA (pH 8.0) (Church and Gilbert, 1984). The membranes were washed in 20 mM NaH2PO4-H3PO4 (pH 7.2) and 1% (w/v) SDS twice for 10 min at 42 °C and then once for 15 min at 45 °C. Autoradiographic images of the membranes were obtained by using imaging plates and a BAS 2000 system (Fuji Film, Tokyo). Positive colonies were isolated and purified by subsequent rounds of screening at lower colony densities.
DNA sequence analysis
Clones with an approximately 1 kb long insert were picked and sequenced by the dideoxy method using a Thermo SequenaseTM kit (Amersham). The fluorescence was detected and analysed with a DSQ-1000L system (Shimadzu, Kyoto, Japan). The complete nucleotide sequences were determined for both strands of the cDNA. One of the longest clones was named HvAPT1.
Total RNA isolation and Northern analysis
Total RNA isolation followed the method of Logmann et al. (Logmann et al., 1987). Total RNA (10 or 15 µg) was separated on a 1.2% (w/v) agarose gel containing 5% (v/v) formaldehyde and blotted onto a nylon membrane (Hybond-N+, Amersham). Hybridization was performed using the method described above. A 220 bp PCR fragment located downstream from the open reading frame (ORF) in HvAPT1 (Fig. 1) was labelled as a specific probe for the Northern analysis of HvAPT1.
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Genomic Southern hybridization
Barley genomic DNA was prepared from leaves with cetyltrimethylammonium bromide using the method of Murray and Thompson (Murray and Thompson, 1980). The DNA was digested with BamHI, EcoRI, or HindIII, separated on a 0.8% (w/v) agarose gel (30 µg per lane), and alkali-transferred onto a nylon membrane (Hybond-N+, Amersham). The membrane was hybridized with a labelled fragment from HvAPT1 at 65 °C overnight. The hybridization buffer contained 5xSSPE, 4xDenhardt's solution, 10% (w/v) dextran sulphate, and 100 mg ml-1 denatured salmon-sperm DNA. The membrane was washed in buffer containing 2xSSPE and 0.1% (w/v) SDS, once at 50 °C for 10 min and then once at 58 °C for 20 min.
Preparing the extract for assaying APRT activity
The preparation of plant extracts and the assay of APRT activity followed the methods of Moffatt and Somerville and Lee and Moffatt with some modifications (Moffatt and Somerville, 1990; Lee and Moffatt, 1993). All the operations were carried out at 4 °C, including the adjustment of the buffer pH. The following buffers were used to prepare the extract: Buffer A, 50 mM PIPES-KOH (pH 6.8), 5 mM MgCl2, 2 mM (p-amidinophenyl)methanesulphonyl fluoride, 2.5 m M DTT, and 2% (w/v) PVP; Buffer B, 50 mM PIPES-KOH (pH 6.8), and 5 mM MgCl2; Buffer C, 50 mM TRIS-HCl (pH 7.8), 10 mM MgCl2, and 10% (v/v) glycerol. The sample, 2 g fresh weight, was thoroughly homogenized in liquid nitrogen and then suspended in 8 ml of Buffer A. The homogenate was centrifuged at 10 000 g for 15 min. Solid (NH4)2SO4 was added gradually to the supernatant to a concentration of 35% (w/v). The pH was adjusted to 7.4 with 1 M KOH and the stabilized supernatant was heated to 58 °C for 5 min and cooled to 4 °C for 5 min. This heated extract was centrifuged at 23 500 g for 10 min to remove denatured proteins. The (NH4)2SO4 concentration of the supernatant was increased to 75% (w/v) and it was stirred gently for 1 h. The protein precipitate was collected by centrifugation at 12 000 g for 30 min and the pellet was resuspended in 0.5 ml of Buffer B. The insoluble debris was removed by centrifugation at 10 000 g for 10 min. As a final step, to prepare the extract for the APRT activity assay, the extract was desalted using a Sephadex G-25 column (Pharmacia Biotech) that had been equilibrated with Buffer C. When this extract was stored in Buffer C at -80 °C, the APRT activity remained stable for over 1 month.
Assay of APRT activity
The protein concentration in the extract described above was determined using the method of Bradford (Bradford, 1976). The activity assay was performed at 37 °C for 5 min and 2 µl of diluted extract containing 0.4–2.5 µg of protein were used for every assay. The reaction mixture was composed of 50 mM TRIS-HCl (pH 7.4), 5 mM MgCl2, 15 mM NaF, 10 mM NaN3, 1 mM phosphoribosyl pyrophosphate (PRPP), 2.3 mg ml-1 bovine serum albumin, and 100 µM [8-14C]adenine (2.0 MBq µmol-1, NEN). The reaction was started by adding the sample to 50 µl of reaction mixture, pre-warmed to 37 °C. After incubation at 37 °C for 5 min, the reaction was terminated by the addition of 500 µl of ice-cold stop solution, containing 50 mM sodium acetate (pH 5.0) and 2 mM K2HPO4. The addition of 50 µl of ice-cold 1 M LaCl3 precipitated the reaction product after 30 min on ice. The precipitate was collected by filtration through a 0.65 µm filter (Ultrafree, Millipore). The membrane was washed three times with ice-cold stop solution and its radioactivity was measured by liquid scintillation counting. As a control, 2 µl of Buffer C was used instead of the extract to assay the amount of unreacted [14C]adenine remaining on the filter membranes.
Construction of plasmid HvAPT1-sGFP
Plasmid pUC18 containing the construct, cauliflower mosaic virus (CaMV) 35S promoter-sGFP(S65T)-NOS 3', was kindly provided by Dr Yasuo Niwa (University of Shizuoka). The construct had SalI and NcoI sites on the 3' side of the CaMV 35S promoter. the NcoI site ‘CCATGG’ included the initiation codon for sGFP (Chiu et al., 1996). The ORF of HvAPT1 was amplified by PCR. The sense primer, 5'-AGACCTCGAGTCGACATGGCATCCGACGGGCGCGTGGAGCGG, had XhoI and SalI sites and the antisense primer, 5'-CGCAGAATTCCATGGCTACTGATTCGTCTGCCTGCACAGGAC, had EcoRI and NcoI sites. In the PCR product, the stop codon ‘TGA’ in HvAPT1 was changed to ‘GCC’ by the antisense primer. The PCR product was digested by XhoI and EcoRI, subcloned into pBluescriptII SK-, and then sequenced to confirm that any mutations, including frame shift mutations, had not been caused by PCR. The HvAPT1 ORF fragment was digested from pBluescriptII SK- by SalI and NcoI and inserted into pUC18 containing the sGFP construct. The resulting construct was CaMV 35S promoter-(HvAPT1 ORF-sGFP)-NOS 3'. For analysis of sGFP localization, pUC18 containing sGFP was used as a control.
Bombardment of tobacco BY-2 cells
Tobacco BY-2 cells (Nagata et al., 1992) were grown in 100 ml NT medium in 300 ml flasks (Newman et al., 1993), and subcultured weekly by transferring 1.5 ml stationary culture to fresh medium. Appropriate amounts of culture medium containing 3–4 d subcultured cells were poured onto a piece of sterilized filter paper (3MM paper, Whatman) on a Petri dish of MS medium solidified with 2% Gellan Gum. The filter paper supporting the cells was air-dried until excess water from the medium had evaporated.
Gold particle bombardment was performed using a helium-driven particle accelerator (PDS-1000/He, BioRad) with all basic settings as the manufacturer's recommendations. Particles with a diameter of 1.0 µm were coated with expression constructs and prepared for bombardment according to the manufacturer's protocol. Plated BY-2 cells were placed under the stopping screen at a distance of 8 cm and bombarded in a vacuum of 28 inches of mercury using a helium pressure of 1550 psi to accelerate the macrocarrier. Bom barded cells were kept in the dark at 26 °C for 4–24 h before analysis.
Confocal microscopy
Bombarded BY-2 cells were mounted in NT medium containing 2% SeaPlaque GTG Agarose (TaKaRa, Tokyo) under glass coverslips. The specimens were examined using a LSM510 laser-scanning confocal microscope equipped with an argon laser and a FITC filter set (Carl Zeiss).
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Results |
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Isolation and sequence analysis of protein ‘C’
Proteins prepared from Fe-sufficient and Fe-deficient barley roots produced different patterns with 2D-PAGE. One spot (20 kDa at pI 5.2) appeared at higher concentrations in Fe-deficient roots than in Fe-sufficient roots and was named protein ‘C’ (Suzuki et al., 1998
). Since the N-terminus of protein ‘C’ was blocked, it was transferred onto a PVDF membrane and digested with either cyanogen bromide or lysylendopeptidase. The resulting peptides were used to derive individual partial sequences. The obtained sequences were ‘KLPGEVISEEYSLEYGTDKI’ and ‘HVGAVQPNDRVLIVDDLIAT’ (Fig. 1
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cDNA cloning from a specific Fe-deficient library
A degenerate primer was designed for PCR using partial amino acid sequences (Fig. 1). A 550 bp long fragment was amplified by PCR and used as a probe to screen a cDNA library. A 975 bp long cDNA containing 180 bp upstream of the first initiation codon was isolated from a cDNA library made from Fe-deficient barley roots. Since the deduced amino acid sequence of the cDNA was highly homologous to adenine phosphoribosyltransferase (APRT; EC 2.4.2.7), especially with respect to plant enzymes, it has been called the HvAPT1 ( Hordeum vulgare aprt) gene. The deduced HvAPT1 protein contains 181 amino acids (Fig. 1). The calculated molecular mass (19.6 kDa and pI 5.0) is very close to that of protein ‘C’ previously identified by 2D-PAGE.
HvAPT1 was highly homologous to wheat APRT, as shown in Fig. 2. The deduced amino acid sequences of HvAPT1 and the aprt gene from wheat differed at only two amino acid residues. The identity between the full-length nucleotide sequences of the two genes was 96%. The homology between the deduced HvAPT1 protein and other APRTs from Escherichia coli (GenBank accession no. M14040), Mus musculus (GenBank accession no. M11310), Drosophila melanogaster (GenBank accession no. L06280), and Leishmania tarentolae (GenBank accession no. AF060886) was 53%, 46%, 44%, and 37%, respectively. APRT binds adenine and PRPP as substrates and the binding domains for these substrates (de Boer and Glickman, 1991) are highly conserved in plant species (Fig. 2).
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HvAPT1 gene expression with changing Fe availability
A 220 bp of 3' untranslated region of HvAPT1 was used as a specific probe for Northern hybridization (Fig. 1). In barley leaves, the HvAPT1 mRNA level was nearly equivalent, irrespective of Fe supply (Fig. 3A). The HvAPT1 expression in Fe-deficient barley roots was much greater than in Fe-sufficient roots. HvAPT1 expression in roots was examined while varying the availability of Fe to the plants. The HvAPT1 mRNA in roots increased after 1 d of Fe deficiency, reached a maximum level after 4–7 d, and then decreased slightly to a stable level (Fig. 3B). When Fe(III) was supplied to plants suffering from 3 weeks of severe Fe shortage, the HvAPT1 expression decreased to the level in normal Fe-sufficient roots (+Fe control in Fig. 3C) within a few days (Fig. 3C).
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APRT activity during Fe deficiency
The enzymatic activity of APRT in Fe-deficient barley roots was several times greater than that of Fe-sufficient barley roots (Fig. 4). In contrast, there were no significant differences in the APRT activities of leaves between Fe-deficient and Fe-sufficient barley plants. A remarkable increase of APRT activity in Fe-deficient roots was also observed in other graminaceous plants (Table 1). APRT activity in Fe-deficient roots was 8.5 times greater than that of Fe-sufficient roots in rye, 3.8 times greater in maize, and 4.8 times greater in rice. In contrast, APRT activity in Fe-deficient roots was only 1.3 times greater than that in Fe-sufficient roots of tobacco, that does not produce MAs.
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Southern hybridization analysis of HvAPT1
Digestion with BamHI produced two fragments that hybridized with the probe; digestion with EcoRI produced one fragment; and digestion with HindIII produced three fragments, although one of them showed only a weak signal (Fig. 5). Since none of these restriction enzyme sites are included in the fragment used as the Southern hybridization probe, it was concluded that at least two copies of the APRT gene exist in the barley genome.
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Localization of the HvAPT1-sGFP fusion protein in BY-2 cells
The HvAPT1 protein fused to the N-terminus of sGFP was transiently expressed under the control of the CaMV 35S promoter in tobacco BY-2 cells. Green fluorescence of sGFP in living BY-2 cells was viewed by a confocal microscopy. As shown in Fig. 6A, the fluorescence of HvAPT1-sGFP fusion protein in a living BY-2 cell was homogeneously distributed in the cytoplasm, including the trans-vacuolar cytoplasmic strands, but not in specific organelles. Accumulation of fluorescence was also observed within the nucleus and the nucleolus was seen in the nucleus as a fluorescence-free space (Fig. 6A). When sGFP alone was expressed (Fig. 6B), the intensity of fluorescence was equivalent in the cytoplasm and the nucleus in concurrence with the distribution of HvAPT1-sGFP fusion protein although the intensity of fluorescence was always higher than that of HvAPT1-sGFP. Thus, the HvAPT1-sGFP fusion protein was thought to be distributed within the cytoplasm of BY-2 cells.
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Discussion |
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Several proteins accumulated specifically in the roots of Fe-deficient barley, and one of these was named protein ‘C’ (Suzuki et al., 1998
). Based on partial amino acid sequences, a cDNA, HvAPT1, was cloned whose deduced amino acid sequence was highly homologous to plant APRTs. APRT is an enzyme that converts adenine into AMP in one step. This reaction is thought to be the main salvage pathway for adenine in higher plants and APRT is counted among housekeeping enzymes.
Northern hybridization analysis showed that more HvAPT1 was expressed in roots than in leaves and that the expression in roots was induced by Fe deficiency, which did not affect expression in leaves (Fig. 3A). Furthermore, the HvAPT1 expression in roots changed when the Fe availability was altered (Fig. 3B, C). Depletion of Fe increased the expression of HvAPT1, and supply of Fe rapidly decreased HvAPT1 expression. The induced HvAPT1 expression in roots was consistent with the appearance of protein ‘C’ from Fe-deficient roots on 2D-PAGE gels.
Although the HvAPT1 mRNA levels in barley leaves were very low (Fig. 3A), the APRT activity was not (Fig. 4), suggesting the presence of another aprt gene in barley that is expressed in leaves. Southern analysis of the barley genome also indicated the existence of the second aprt gene (Fig. 5). Ashihara and Ukaji detected APRT activity in extracts from chloroplasts in spinach leaves (Ashihara and Ukaji, 1985). It is conceivable that two different types of aprt gene are present in plants; one that is expressed mainly in roots and another that is expressed mainly in leaves. The authors have tried to isolate the other postulated barley aprt gene to examine its relationship to Fe deficiency. Several different cDNA libraries have been used, but so far have not yet been successful. The difficulties in cloning a cDNA encoding an isozyme might be because the mRNA used for the libraries was not a suitable source.
Two aprt genes, ATapt1 and ATapt2, were isolated from Arabidopsis thaliana (Moffatt et al., 1992; Schnorr et al., 1996). ATapt2 is expressed in roots, stems and flower meristems, but not in leaves, while ATapt1 is expressed in leaves. This result also suggests that another aprt gene might exist in barley in addition to HvAPT1. In addition to the tissue-specific expression of ATapt2, the enzymes encoded by ATapt1 and ATapt2 show different affinities for adenine and benzyladenine. Two APRT isozymes with differing affinity for cytokinin were also observed in different tomato tissues (Burch and Stuchbury, 1986). Although Moffatt et al. demonstrated the in vivo ability of APRT to convert cytokinin bases to their phosphoadenylated forms in A. thaliana, the role of APRT in the metabolism of cytokinin bases is not completely clear (Moffatt et al., 1991).
The total APRT activity in Fe-deficient roots was measured in three graminaceous plants and one non-graminaceous plant, in addition to barley plants. The activity in Fe-deficient barley roots was 6.8 times higher than in Fe-sufficient roots (Fig. 4; Table 1), a finding consistent with the results of Northern analysis (Fig. 3A). The induced activity of APRT in Fe-deficient roots was also observed in other graminaceous plants including rye, maize and rice (Table 1). The expression of an aprt gene might also be induced in roots of these graminaceous plants, as the expression of HvAPT1 was induced in Fe-deficient barley roots. In contrast, the APRT activity did not significantly increase in Fe-deficient roots of a non-graminaceous plant, tobacco (Table 1). These results indicate that the distinct increase in APRT activity in roots during Fe deficiency is specific to graminaceous plants and does not occur in non-graminaceous plants. Graminaceous plants synthesize and secrete MAs and the amount of secreted MAs increases under Fe-deficient conditions. In contrast, non-graminaceous plants adapt a different strategy (Strategy-I) to acquire Fe and neither synthesize nor secrete MAs under Fe-deficient conditions (Marschner et al., 1986). Therefore, the great increase in APRT activity during Fe deficiency in graminaceous plants appears to be closely related to the biosynthesis of MAs. As shown in Fig. 7, the biosynthesis of MAs begins when ATP activates a Met molecule to form S-adenosylmethionine (SAM), a compound involved in many biochemical reactions. The aminobutyrate groups of three SAMs are consumed to form one nicotianamine molecule, and then MAs are biosynthesized via an unstable keto form (Shojima et al., 1990). At the same time, 5'-methylthioadenosine (MTA) is produced as the remainder of the SAM molecule after nicotianamine synthesis (Fig. 7). The concentration of Met in barley roots is very low (Mori et al., 1991), although the production of MAs under Fe-deficient conditions is very high. MAs production under Fe-deficient conditions can reach 1% of the dry weight of roots per day (Takagi, 1984). Ma et al. (Ma et al., 1995) found that the Met supply for MAs is mediated by the Met cycle (Fig. 7), as occurs in ethylene and polyamine production (Miyazaki and Yang, 1987). This finding was supported by a previous study, which showed that [11C]Met was not transported from the top of a leaf to the roots (Nakanishi et al., 1999). the Met used in MAs biosynthesis in roots may not come from the leaves but is synthesized in the roots themselves through the Met cycle. Furthermore, a gene named IDI1 has recently been identified from Fe-deficient barley, which presumably functions in the Met cycle to convert 5-methylthioribose-1-phosphate (MTR-1-P) to 2-keto-4-methylthiobutyrate (KMB) (Fig. 7; Yamaguchi et al., 2000). The fact that the expression of IDI1 was induced in Fe-deficient roots indicates that the Met cycle works vigorously in barley roots under Fe deficiency.
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In the Met cycle, the three molecules of MTA that result from nicotianamine production are metabolized into 5-methylthioribose (MTR) and, in turn, regenerated into Met. At this step of MTR formation, adenine, which is derived from ATP for the S-adenylation of Met, is released from the cycle (Fig. 7
). Giovanelli et al. reported that in Lemna adenine derived from MTA is efficiently salvaged for the synthesis of adenine nucleotide such as AMP, ADP, and ATP (Giovanelli et al., 1983). Generally, APRT is a housekeeping enzyme which converts free adenine base to AMP in one step. In contrast, the expression of HvAPT1 changes with the availability of Fe in the plant, suggesting that the APRT from HvAPT1 may function to salvage the large amount of adenine released from MTA during MAs biosynthesis. AMP synthesized by APRT would finally be converted to ATP, which again takes part in the Met cycle through S-adenylation of Met or phosphorylation of MTR. Considering the induction of APRT activity in other graminaceous plant roots by Fe deficiency (Table 1), APRT is probably connected to physiological conditions of Fe nutrition of graminaceous plants.
The authors have reported on the formate dehydrogenase gene (Fdh), which is also induced by Fe deficiency in barley roots (Suzuki et al., 1998). This induction of Fdh gene expression was delayed and thought to be a result of anaerobic stress caused by Fe deficiency. In the Met cycle, formate is released as well as adenine (Fig. 7). Therefore, induction of the Fdh gene by Fe deficiency treatment might also be initiated by the formate that is released from MTR-1-P in the Met cycle.
The details of the regulation of the whole Met cycle are not yet known. In general, there are several SAM synthetase genes in each plant. This enzyme is responsible for Met activation (Fig. 7). Three cDNAs encoding SAM synthetase from barley have been cloned to examine their relationship to Fe deficiency (DDBJ accession nos. D63835, D85237, D85238). However, expression of these SAM synthetase genes was not induced by Fe deficiency (Takizawa et al., 1996). There might be another SAM synthetase gene in barley, whose expression is induced by Fe deficiency, since the expression of IDI1, a putative gene for the Met cycle, was increased dramatically in Fe-deficient barley roots (Yamaguchi et al., 2000). In the biosynthetic pathway of MAs from Met, the enzymatic activity of NAS, which combines three SAM molecules, is induced by Fe deficiency and suppressed by Fe supply. Thus, Fe directly regulates the expression of nas genes (Higuchi et al., 1999). Fe also regulates the naat expression directly (Takahashi et al., 1999). The authors have now shown that Fe involves the expression of HvAPT1 in barley roots and postulate that Fe-deficiency induced expression of APRT is linked to MAs production in grasses.
Based on the postulated function of APRT in MAs production, the cellular localization of HvAPT1 protein was investigated using a transient expression system for sGFP fusion proteins. The HvAPT1-sGFP fusion protein was distributed within the cytoplasm of living tobacco BY-2 cells (Fig. 6). This localization is presumably the same as that in cells of graminaceous plants, since APRT is one of the housekeeping enzymes of higher plants. The HvAPT1 protein might be present in the cytoplasm of barley cells. Previously, the appearance of specific vesicles derived from the rough endoplasmic reticulum (rER) in the root cells of Fe-deficient barley before sunrise, just before the diurnal secretion of mugineic acids (Nishizawa and Mori, 1987) has been reported. In addition, from the analyses of their amino acid sequences, NAS and NAAT, the key enzymes of MAs biosynthesis, are both predicted to be sorted in the rER (Higuchi et al., 1999; Takahashi et al., 1999). It is postulated that MAs are produced in the particular vesicles and stored until they are secreted. Localization of the HvAPT1 protein in the cytoplasm suggests that substrates for NAS are produced in the cytoplasm. That is to say, nicotianamine may be synthesized in the particular vesicles using the aminobutyrate group of SAM supplied from the cytoplasm. To locate the site of MAs synthesis and SAM generation by the Met cycle precisely, the intracellular localization of NAS, NAAT, SAM synthetase, IDI1, and APRT should be investigated by immunocytochemistry in the future.
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Acknowledgments |
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We thank Mrs Kazuko Okamoto of the University of Tokyo for the technical assistant of analysis of amino acid sequencing.
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Notes |
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3 To whom correspondence should be addressed. Fax: +81 3 5684 4822. E-mail: asmori{at}mail.ecc.u-tokyo.ac.jp
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Abbreviations |
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APRT, adenine phosphoribosyltransferase; IDI1, iron deficiency induced cDNA 1; MAs, mugineic acid family phytosiderophores; NAAT, nicotianamine aminotransferase; NAS, nicotianamine synthase; sGFP, synthetic green fluorescent protein.
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