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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Phytic Acid Synthesis and Vacuolar Accumulation in Suspension-Cultured Cells of Catharanthus roseus Induced by High Concentration of Inorganic Phosphate and Cations^1,[w]

Japan Society for the Promotion of Science, Tokyo 102–8471, Japan (N.M.); Department of Biology, Faculty of Science, Kobe University, Kobe 657–8501, Japan (N.M., M.O., T.M.); Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo 113–0027, Japan (M.O., T.M.); Technical Department, Nippon Dionex K.K., Osaka 532–0011, Japan (Y.S.); Department of Chemistry, Pohang University of Science and Technology, Pohang 790–784, Korea (Y.-U.K., S.-K.C.); Department of Chemistry, New York University, New York, New York 10003 (Y.-T.C.); Department of Preventive Medicine and Environmental Health, Osaka City University, Medical School, Osaka 545–8585, Japan (Y.I.); School of Earth and Environmental Sciences, University of Adelaide, South Australia 5005, Australia (R.J.R.); and Department of Life Science, Graduate School of Science, University of Hyogo, Harima Science Garden City, Hyogo 678–1297, Japan (H.Y.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
We have established a new system for studying phytic acid, myo-inositol hexakisphosphate (InsP₆) synthesis in suspension-cultured cells of Catharanthus. InsP₆ and other intermediates of myo-inositol (Ins) phosphate metabolism were measured using an ion chromatography method. The detection limit for InsP₆ was less than 50 nM, which was sufficient to analyze Ins phosphates in living cells. Synthesis of Ins phosphates was induced by incubation in high inorganic phosphate medium. InsP₆ was mainly accumulated in vacuoles and was enhanced when cells were grown in high concentration of inorganic phosphates with the cations K⁺, Ca²⁺, or Zn²⁺. However, there was a strong tendency for InsP₆ to accumulate in the vacuole in the presence of Ca²⁺ and in nonvacuolar compartments when supplied with Zn²⁺, possibly due to precipitation of InsP₆ with Zn²⁺ in the cytosol. A vesicle transport inhibitor, brefeldin A, stimulated InsP₆ accumulation. The amounts of both Ins(3)P₁ myo-inositol monophosphate synthase, a key enzyme for InsP₆ synthesis, and Ins(1,4,5)P₃ kinase were unrelated to the level of accumulation of InsP₆. The mechanisms for InsP₆ synthesis and localization into vacuoles in plant cells are discussed.

Research into Ins phosphates in plants first concentrated on measuring levels in seeds, mainly of InsP₆ (Holt, 1955; Asada et al., 1969; Griffiths and Thomas, 1981; Raboy et al., 1984), and on the investigation of reaction kinetics of the enzymes involved in synthesis of InsP₆ (Phillippy et al., 1994; Phillippy, 1998; Brearley and Hanke, 2000; Stevenson-Paulik et al., 2002). From a human nutrition perspective, high InsP₆ in grain is considered undesirable due to its binding of essential micronutrient metals. Low-InsP₆ mutants have been isolated from barley (Hordeum vulgare; Hatzack et al., 2000; Dorsch et al., 2003), maize (Zea mays; Raboy et al., 2000), rice (Oryza sativa; Larson et al., 2000), and soybean (Glycine max; Wilcox et al., 2000). A maize low-phytic acid mutant lpa2 arose by mutation in an Ins(1,3,4)P₃ 5/6 kinase gene (Shi et al., 2003), which caused the InsP₆ content to be reduced by approximately 30%, and the Pi to increase about 3-fold. Another mutant in maize (lpa241) showed approximately 90% reduction of InsP₆, 10-fold increase in seed-free phosphate content, and reduction of Ins(3)P₁ synthase gene expression (Pilu et al., 2003).

Many aspects of the synthesis of InsP₆ are also well investigated. The early step of InsP₆ synthesis is the conversion of Glu-6-P to Ins(3)P₁ mediated by Ins(3)P₁ myo-inositol monophosphate synthase (MIPS). The latter steps are consistent with sequential phosphorylations of soluble Ins phosphates mediated by several kinases and phospholipase C-dependent conversion of phosphatidyl inositol phosphate intermediates to Ins(1,4,5)P₃ (Loewus and Murthy, 2000; Raboy, 2003). MIPS gene expression is suppressed by addition of Ins (Johnson and Sussex, 1995).

In spite of these efforts, the understanding of the factors that regulate synthesis of InsP₆ is patchy. During seed maturation, the accumulation of InsP₆ in seeds was strongly affected by the level of Pi in the culture solution (Asada et al., 1969; Raboy and Dickinson, 1984). Yoshida et al. (1999) showed that strong signals of transcript of a rice MIPS gene (RINO1) were detected between 4 and 7 d after anthesis in scutellum and aleurone layer, coinciding with the appearance of phytin. They subsequently reported that treatment of rice cultured cells with abscisic acid and Suc together resulted in much higher levels of MIPS transcript accumulation, suggesting a synergistic induction of the MIPS gene (Yoshida et al., 2002). It was recently shown that MIPS activity is widely distributed in intracellular compartments, including membrane-bound organelles and cell walls, as well as cytoplasm (Lackey et al., 2003), but the site(s) of InsP₆ synthesis has not been established, nor is it known how InsP₆ is transported to the vacuole, which becomes globoid in seeds. Most of the InsP₆ in castor bean (Ricinus communis) seeds is bound to Ca²⁺ and Mg²⁺ and accumulated in globoid in protein storage vacuoles (Greenwood and Bewley, 1984). In developing seeds of Arabidopsis (Arabidopsis thaliana), embryo globoids contained Mg²⁺, K⁺, and Ca²⁺, the charazal endoplasmic reticulum (ER) showed high level of Mn²⁺, and the charazal vacuoles contained zinc phytate (Otegui et al., 2002).

Here, we report the development of an experimental system for the in vivo investigation of the dynamics of synthesis and compartmentation of InsP₆ using suspension-cultured cells of Catharanthus.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Induction of InsP₆ Accumulation in Suspension-Cultured Cells

Although most interest in InsP₆ is focused on developing seeds, it is quite difficult to control and synchronize the development experimentally. As an alternative, we turned to suspension-cultured Catharanthus cells. When grown in Murashige and Skoog (MS) medium with 1.25 mM Pi, the cells depleted Pi in the medium and were effectively starved after 7 d (low-Pi cells) and contained negligible amounts of InsP₆ (Fig. 1, A and B). If the medium was supplemented with 7.5 mM Pi at day 3 and day 5, cells then accumulated high concentrations of both Pi and InsP₆ (high-Pi cells). While the concentration of InsP₆ continued to increase over the 7 d period (to 165.5 nmol g fresh weight [FW]^–1), the concentration of Pi peaked after 4 d (36.8 µmol g FW^–1) and thereafter remained constant. In addition to InsP₆, various other isomers of myo-inositol tetrakisphosphate (InsP₄) and myo-inositol pentakisphosphate (InsP₅) were detected in high-Pi cells (Fig. 1C), but myo-inositol monophosphate (InsP₁), myo-inositol bisphosphate (InsP₂), and myo-inositol trisphosphate (InsP₃) were either absent or below the detection limit. InsP₆ and other intermediates of Ins phosphate metabolism were measured using an ion chromatography method (see "Materials and Methods" and supplemental data).

View larger version (22K): [in this window] [in a new window]   Figure 1. Changes in levels of InsP6 and Pi in suspension-cultured Catharanthus cells grown with normal and high concentration of Pi. Low-Pi cell (represented by triangles and broken line) were grown with a normal MS salt containing 1.25 mM Pi for 7 d after transfer to fresh medium. High-Pi cells (represented by circles and straight line) were grown in MS salt with 2 times supplementation of 7.5 mM of Pi at days 3 and 5 (indicated by arrows). The 0, 2, 4, 6, and 7 d cells were harvested and homogenized in 2.4% (w/v) HCl. InsP6 (A) and Pi (B) in the extracts were measured by ion chromatography. C, Assignment of Ins phosphates in high-Pi cells. Each peak was assigned as follows: 1, InsP6; 2, Ins(2,4,5,6)P4; 3, D/L-Ins(1,2,4,5,6)P5; 4, Ins(1,2,3,4,6)P5; 5, D/L-Ins(1,2,3,4,5)P5; 6, D/L-Ins(1,4,5,6)P4; 7, D/L-Ins(1,3,4,5)P4; 8, D/L-Ins(1,2,4,5)P4. D, Changes in levels of InsP6 in suspension-cultured Arabidopsis cells grown with normal and high concentration of Pi. Low-Pi cell (represented by diamonds and broken line) were grown with a modified MS salt containing 3.75 mM Pi for 7 d after transfer to fresh medium. High-Pi cells (represented by squares and straight line) were grown in modified MS salt with 2 times supplementation of 7.5 mM of Pi at days 3 and 5 (indicated by arrows). The 0, 1, 3, 5, and 7 d cells were harvested and homogenized in 2.4% (w/v) HCl.

Cellular Localization of Ins Phosphates

The subcellular location of Ins phosphates in the cultured cells was investigated by comparing the profiles of Ins phosphates in protoplasts and in vacuoles isolated from the protoplasts. The measured contents were normalized using the activity of -mannosidase, a vacuole-specific marker enzyme.

High-Pi cells were found to contain more than half of the InsP₆ in their vacuoles (Fig. 2A), while vacuoles from low-Pi cells accumulated very low levels of InsP₆ (Fig. 2B). Low levels of InsP₄s and InsP₅s were found in protoplasts of high-Pi cells. These peaks were confirmed to be Ins phosphates by the addition of phytase (phytase from Aspergillus ficuum, Sigma, St. Louis; Fig. 2C).

View larger version (17K): [in this window] [in a new window]   Figure 2. A, Distribution of Ins phosphates in protoplasts and vacuoles from high-Pi cells. Double asterisks indicate mixtures of InsP4s and/or InsP5s shown in Figure 1C. B, Absence of Ins phosphates in protoplasts and vacuoles from low-Pi cells. C, Changes in levels of Ins phosphates following addition of phytase to extracts. The asterisks in A indicate unassigned peaks that were diminished by phytase. The extracts were normalized to 20 mU -mannosidase activity.

In mature dry seeds, InsP₆ is usually bound to K⁺, Ca²⁺, and Mg², forming phytin globoids. Thus, accumulation of InsP₆ might be closely related to storage of cations. The effects of cations on accumulation of InsP₆ were investigated by growing Catharanthus cells in high-Pi medium with Ca²⁺ (80 mM), Mg²⁺ (50 mM), Zn²⁺ (1 mM), or Mn²⁺ (3 mM) for 7 d. Concentrations of divalent cations were selected according to Hirschi et al. (2000). The amount of InsP₆ in cells incubated with Pi plus Ca²⁺ or Zn²⁺ increased markedly compared to that in cells supplied only with Pi, but decreased slightly in cells incubated with Mg²⁺ or Mn²⁺ (Fig. 3A). K⁺ is a major monovalent cation detected in phytin globoids of some species (Otegui et al., 2002). Addition of 125 mM K⁺ in high-Pi increased InsP₆ markedly, but 75 mM K⁺ cells showed levels comparable to that in high-Pi cells (Fig. 3A). The Pi concentration in each treatment was almost unchanged, except for high-Pi + Ca²⁺ cells in which the Pi concentration increased 2-fold (Fig. 3B).

View larger version (24K): [in this window] [in a new window]   Figure 3. Effect of metal cations on InsP6 synthesis induced by high concentration of Pi. Catharanthus cells were grown under high-Pi with 50 mM MgCl2, 80 mM CaCl2, 1 mM ZnCl2, 3 mM MnCl2, 75 mM KCl, or 125 mM KCl. The 7-d cells were harvested and homogenized in 2.4% (w/v) HCl. InsP6 (A) and Pi (B) in the extracts were measured by ion chromatography. Each value is represented as percentage of relative content to high-Pi cells. Data are average of at least three experiments.

View larger version (18K): [in this window] [in a new window]   Figure 4. Effect of metal cations on accumulation of InsP6 in vacuoles. Protoplasts and vacuoles were isolated from 7-d Catharanthus cells grown under high-Pi with 50 mM MgCl2, 80 mM CaCl2, 1 mM ZnCl2, 3 mM MnCl2, or 125 mM KCl. The amounts of InsP6 in protoplasts and vacuoles were normalized to 20 mU of -mannosidase activity to enable comparisons. Data show typical chromatograms in more than three independent experiments. The means of InsP6 amounts occupied in vacuoles versus protoplasts were shown in the right.

The level of MIPS in Catharanthus was investigated with a specific antibody against a 62-kD recombinant Arabidopsis MIPS (At4g39800) protein. The antibody showed cross reactivity to a 56-kD Catharanthus protein (Fig. 5A), whose expression was reduced by addition of Ins in a dose-dependent manner (Fig. 5A, lanes 8 and 9). The Catharanthus MIPS homolog was found to be constitutively expressed in 7-d-old cells in all of the treatments examined here (Fig. 5A, lanes 2–7). Likewise, K⁺ did not alter the level of MIPS (data not shown). Thus, increase of InsP₆ content and/or the promotive effect by metal cations were not caused by induction of MIPS.

View larger version (39K): [in this window] [in a new window]   Figure 5. Immunoblot analyses for (A) MIPS and (B) Ins(1,4,5)P3 kinase (Ipk2) in Catharanthus cells grown under various conditions. Immunoblots with preimmune (A and B, lane 1), anti-MIPS (A, lanes 2–12), or anti-Ipk2 (B, lanes 2–9) are shown. The lanes show total protein (80 µg) from protoplasts of low-Pi (A and B, lane 2), high-Pi (A and B, lane 3), high-Pi + 80 mM CaCl2 (A and B, lane 4), high-Pi + 1 mM ZnCl2 (A and B, lane 5), high-Pi + 50 mM MgCl2 (A and B, lane 6), or high-Pi + 3 mM MnCl2 (A and B, lane 7); total proteins (80 µg) from cells of +10 mM Ins (A and B, lane 8), +50 mM Ins (A and B, lane 9); total proteins (10 µg) from mature leaves (A, lane 10) and immature seeds (A, lane 11) of Arabidopsis; recombinant MIPS protein (0.08 µg; A, lane 12); or recombinant thioredoxin-AtIpk2 fusion protein (0.2 µg; B, lane 10).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

InsP₆ Synthesis and Accumulation in the Vacuole

The use of suspension-cultured Catharanthus cells provides an experimental system for investigating InsP₆ synthesis in vivo. Accumulation of InsP₆ in the vacuole was easily inducible in these cells when grown with high concentration of Pi (Fig. 2A). Catharanthus cells can grow in the medium (1.25 mM Pi) without Ins (see "Materials and Methods"). By contrast, Arabidopsis cells need the medium containing Ins and higher Pi (3.75 mM). These differences may influence levels of InsP₆ synthesis in both cells (Fig. 1, A and D). Thus, we concluded that Catharanthus cells are more suitable material for investigating InsP₆ synthesis than Arabidopsis cells.

Various isomers of InsP₄ and InsP₅ were also detected in Catharanthus cells (Fig. 1C). Part of them was also detected in vacuoles (Figs. 2A and 4). However, these may be degraded products of InsP₆. InsP₁ to InsP₃ were not detected, which may indicate their rapid sequential phosphorylation into higher Ins phosphates without the accumulation of intermediates.

The actual site of InsP₆ synthesis is unknown, although the requirement for ATP for the phosphorylation steps from InsP₁ to InsP₆ strongly points to a cytosolic location. Indeed, MIPS localizes to the whole cytoplasm in Phaseolus vulgaris (Lackey et al., 2003). The questions that need solutions are (1) what regulates the synthesis of InsP₆, and (2) how is it transported to the vacuole? In relation to the first question, there is clearly some association between cellular Pi and InsP₆ (Fig. 1) and, as we have shown, between InsP₆ and certain metals (Fig. 3). Ca²⁺, Zn²⁺, and Mg²⁺ are known to form insoluble precipitates with InsP₆ (Urbano et al., 2000), and therefore compartmentation of these metals following uptake will be important. At a high concentration (125 mM), K⁺ induced an increase in InsP₆ (Fig. 3), but this might be the result of osmotic stress, and the synthesis of InsP₆ may be to provide a compatible osmotic solute to counter this stress. The concentration of Ca²⁺ in the cytosol is maintained at submicromolar levels, although Ca²⁺ can reach much higher levels in the vacuole (our data suggest 2–3 mM in Catharanthus; data not shown). It is therefore not surprising that under high Ca²⁺ conditions, almost all InsP₆ was detected in the vacuole (Fig. 4). The finding that under high Zn²⁺ supply most InsP₆ occurred in the cytosol and that a significant proportion could be recovered in an insoluble fraction (Fig. 4; Table I) seems to indicate that the concentration of Zn²⁺ in the cytosol under these conditions is high enough to induce precipitation. An in vitro analysis showed that 200 µM InsP₆ was completely precipitated by addition of 1 mM ZnCl₂ or CaCl₂ at pH 7.2 (data not shown). This could possibly be due to the reduction in InsP₆ free acid caused by increased precipitation stimulating de novo synthesis of InsP₆ via sequential enzymatic equilibrium. Recently, cellular functions for InsP₆ in plants other than storage of Pi have been proposed, such as an abscisic acid-induced Ca²⁺ release in guard cells (Lemtiti-Chieh et al., 2003) or a relationship to turion formation in duckweed (Spirodela polyrrhiza; Flores and Smart, 2000). Under these circumstances, it may be necessary to compensate for the loss of InsP₆ due to chelation in order to maintain an appropriate level of free InsP₆ to regulate these other activities.

Metabolic Regulation of Ins Phosphates Synthesis

In Arabidopsis, three genes of MIPS (At2g22240, At4g39800, and At5g10170) are highly conserved and the amino acid identities are >89%. Since the present antibody is polyclonal, MIPS expressed by genes other than At4g39800 might be detected. Although we do not know how many MIPS genes are in Catharanthus genome, it is likely that the antibody detected most MIPS expressed in suspension-cultured Catharanthus cells. AtIpk2 is localized in nuclei and catalyzes the conversion from D-Ins(1,4,5)P₃ to D-Ins(1,3,4,5,6)P₅ (Xia et al., 2003). We have shown that the level of Ins(1,4,5)P₃ kinase (Ipk2), as well as MIPS (Fig. 5), did not noticeably differ under conditions of either high or low InsP₆ accumulation, and is therefore unlikely to be a candidate for the regulation of synthesis of InsP₆ in the present condition. Addition of Ins suppressed both enzymes (Fig. 5). Suppression of MIPS gene in the presence of Ins has been well known (Johnson and Sussex, 1995), but Ins-dependent suppression of Ipk2 may be the first report. Although in this condition (with Ins and without Pi) InsP₆ was not accumulated, the regulation mechanism of those enzymes by Ins and Pi is a future subject. As yet it is not clear whether expression level of Ins(1,3,4) 5-/6-kinase is very weak or the antibody used here has no cross reactivity to the kinase protein in Catharanthus cells. We should improve the analytical conditions to investigate this kinase in detail.

The mechanism by which InsP₆ is transported to the vacuole remains to be resolved. It has been suggested that InsP₆ may be transported from ER lumen to protein storage vacuoles via a vesicle transport pathway (Greenwood and Bewley, 1984). This hypothesis was partly supported by the brefeldin A experiments (Table II), but the incorporation of InsP₆ into the ER lumen was not demonstrated. Brefeldin A had a strong stimulatory effect on InsP₆ synthesis and its accumulation in the vacuole. This would seem hard to reconcile with the expected inhibition of vesicle transport by brefeldin A, but it is known that brefeldin A has a range of effects on the endomembrane system, some of which (e.g. tubulation of Golgi) may in fact facilitate transfer to the vacuole (Klausner et al., 1992). Wortmannin and monensin also affected InsP₆ synthesis, suggesting phosphoinositide metabolism and membrane transport are also related to InsP₆ synthesis.

In conclusion, the suspension-culture system described here has many advantages for investigating the regulation of synthesis and compartmentation of InsP₆. Additionally, the improvements to the detection system allow quantitative measurements of the key intermediates of InsP₆ metabolism. With the increasing efforts to produce genetically modified plants containing lower levels of Ins phosphates or for environmental phytoremediation (Raboy, 2001; Brinch-Pedersen et al., 2002), these research technologies will be useful.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Ins Phosphates Measurement

For Ins phosphates and Pi measurements, a DX-500 ion chromatography system (Dionex, Osaka) consisting of a gradient pump, a 25-µL sample loop, and a conductivity detector was used as described previously (Baluyot and Hartford, 1996; Sekiguchi et al., 2000). Dionex IonPac AS11 (2 mm i.d. x 250 mm) and IonPac AG11 (2 mm i.d. x 50 mm) columns packed with anion-exchange resin were used as the separation columns. The Dionex ASRS-Ultra anion self-regenerating suppressor was operated in the external water mode at 100 mA. The Dionex PeakNet workstation was used for data processing. A Dionex EG40 eluent generator equipped with an EGC-KOH cartridge was used. A Dionex IonPac ATC-1 (4 mm i.d. x 35 mm), a high-capacity anion-exchange column, was placed at the pump outlet to remove the small amount of dissolved carbon dioxide and carbonate in the deionized water. The current method could separate InsP₁ to InsP₆ in a single gradient elution. Twenty-five microliters of the filtered samples were automatically injected by an autosampler AS-50 (Dionex). The flow-rate was 0.35 mL min^–1 at 35°C. The concentration gradient (5–80 mM KOH) was generated by EG40. The detection limit (S/N = 3) for InsP₆ would be less than 50 nM. Our system could not distinguish enantiomers, but many isomers could be separated among 64 species of Ins phosphates; we could finally separate three of six InsP₁ isomers, three of 15 InsP₂ isomers, eight of 20 InsP₃ isomers, eight of 15 InsP₄ isomers, and three of 6 InsP₅ isomers independently (see supplemental material).

Reagents

InsP₆ was purchased from Sigma. All synthetic isomers of InsP₁s (Chung and Chang, 1996a), InsP₂s (Chung et al., 1998), InsP₃s (Chung et al., 1996), InsP₄s (Chung and Chang, 1995), and InsP₅s (Chung and Chang, 1996b) were synthesized, and their structures and purities were confirmed in Dr. Sung-Kee Chung's laboratory (Pohang University of Science and Technology, Korea).

Plant Materials

Catharanthus roseus L. G. Don cells were cultured in 20 mL of MS medium, pH 6.2, supplemented with 4 µM nicotinic acid, 2.5 µM pyridoxine, 0.3 µM thiamine, 20 µM Gly, 4.5 µM 2,4-dichlorophenoxyacetic acid, and 3% (w/v) Suc (MS medium). Cells were cultured with shaking at 26°C under dim light and transferred to fresh medium every 7 d.

Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 suspension cells were cultured in 20 mL of MS medium, pH 6.2, supplemented with 2.5 mM KH₂PO₄, 1 mM Ins, 16 µM nicotinic acid, 10 µM pyridoxine, 60 µM thiamine, 4.5 µM 2,4-dichlorophenoxyacetic acid, and 3% (w/v) Suc (modified MS medium). Cells were cultured with shaking at 23°C under dim light and transferred to fresh medium every 7 d.

For treatment with high concentrations of Pi, 7.5 mM KH₂PO₄ was added twice (3 and 5 d) during the 7 d culture. For treatment with high-metal cations, 25 mM MgCl₂, 40 mM CaCl₂, 0.5 mM ZnCl₂, 1.5 mM MnCl₂, or 27.5 mM or 52.5 mM KCl were added twice (3 and 5 d) with 7.5 mM Pi in the culture medium. To investigate the effect of various inhibitors on InsP₆ synthesis, 6-d cells cultured in high concentration of Pi were treated for 24 h with 5 mM of Brefeldin A (Wako Pure Chemical Industries, Osaka), 5 mM of wortmannin (Wako), or 2.5 mM of monensin (Sigma). Each chemical was dissolved in dimethyl sulfoxide as stock solutions (x1,000).

To determine Pi and Ins phosphates contents, harvested cells were ground with a mortar and a pestle in liquid nitrogen and homogenized in 2.4% (w/v) HCl. The homogenates were boiled for 10 min and centrifuged at 20,000g for 10 min at 4°C. The supernatant was filtered through a 0.45-µm filter (Ekicro-disc AcroLC, Pall Gelman Laboratory, Tokyo) and was diluted with deionized water, then 25 µL of the filtrate was subjected to ion chromatography. The HCl-extracts were kept for up to 48 h at 37°C, then the samples were measured. The peak area of InsP₆ was not affected by storage for up to 48 h (data not shown).

Isolation of Protoplasts and Vacuoles

Protoplasts and vacuoles were isolated as described previously (Massonneau et al., 2000; Shimaoka et al., 2004). For analysis of contents, protoplasts and vacuoles were freeze-thawed. A part of them was treated with 2.4% (w/v) HCl for measurement of Ins phosphates, as described above.

Assay of -Mannosidase Activity

Freeze-thawed protoplasts and vacuoles were subjected to assay of -mannosidase activity. Two milligrams of p-nitrophenyl--mannopyranoside were dissolved in 1 mL of dimethylformamide (100 x substrate). Each 100 µL of sample was mixed with 400 µL of 1 x substrate diluted by 100 mM sodium-citrate buffer, pH 5.6, otherwise with citrate buffer only. After incubation for 1 h at 37°C, the reaction was stopped by addition of 1 mL of 200 mM Na₂CO₃. Samples were centrifuged at 5,800g for 2 min, and the absorbance of each supernatant was measured at 405 nm. One unit of -mannosidase activity was defined by one absorbance unit at 405 nm.

Measurement of InsP₆ and Pi in Soluble and Insoluble Fraction of Catharanthus Cells

Seven-day-old Catharanthus cells were sonicated (S-250D; Branson, Danbury, CT) in four volumes of buffer A composed of 10 mM Tris-HCl, pH 7.5, with 13% (w/v) Suc. The homogenate was centrifuged at 3,000g for 10 min at 4°C. The centrifuged supernatant and precipitate resuspended in buffer A were collected and subjected to measurement of InsP₆ and Pi as described above.

Measurement of Zinc

Intracellular zinc content was measured with Zincon (Dojindo, Tokyo). Forty microliters of 1 mM Zincon was added into 200 µL of samples diluted in 50 mM Tris-HCl, pH 8.0, and absorbance of the samples was measured immediately at 620 nm.

Preparation for Antibodies against MIPS and Ins(1,4,5)P₃ Kinase

Arabidopsis expressed sequence tag clones (accession nos. AV525103 and AV528014) for an Arabidopsis MIPS gene (At4g39800) and an Arabidopsis Ins(1,4,5)P₃ kinase gene (At5g07370), respectively, were provided from Kazusa DNA Research Institute, Chiba, Japan (http://www.kazusa.or.jp/). For a MIPS gene, two primers, 5'-GAATTCATGTTTATTGAGAGCTTCAAAGTT-3' and 5'-CTCGAGCTTGAACTCCATGATCATGTTGTT-3', were designed on the basis of N-terminal and C-terminal sequences of At4g39800, respectively. The amplified DNA was digested by XhoI and EcoRI and inserted into a XhoI-EcoRI site of pET21a vector (EMD Biosciences, San Diego). The ligated At4g39800-pET21a plasmid was introduced into Escherichia coli BL21(DE3) strain (EMD Biosciences). For an Ins(1,4,5)P₃ kinase gene, two primers, 5'-GGATCCATGCAGCTCAAAGTCCCTGAACAT-3' and 5'-GTCGACCTAAGAATCTGCAGACTCATCTGG-3', were designed on the basis of N-terminal and C-terminal sequences of At5g07370, respectively. The amplified DNA was digested by BamHI and SalI and inserted into the BamHI-SalI site of pET32a vector (EMD Biosciences). The ligated At5g07370-pET32a plasmid was introduced into E. coli BL21(DE3) strain (EMD Biosciences). The recombinant proteins were purified via a 6 x His-tag by using HiTrap Chelating HP column (Amersham Biosciences, Piscataway, NJ) and used as antigens. Specific antisera were provided by Shibayagi (Gunma, Japan).

Immunoblot Analysis

Crude extracts were prepared from Arabidopsis plants and Catharanthus protoplasts according to the following procedures. Immature seeds in green siliques (grown for 4 weeks) and mature leaves (grown for 4 weeks) of Arabidopsis or protoplasts isolated from 7-d-old Catharanthus cells were ground with a mortar and pestle in liquid nitrogen and resuspended with 10 mM Tris-HCl, pH 7.5. All samples were subjected to SDS-PAGE with 7.5% or 10% (w/v) acrylamide gel and electrically transferred to a polyvinylidene difluoride membrane (Bio Craft, Tokyo). The membrane blot was incubated with specific antibodies against Arabidopsis MIPS or Ins(1,4,5)P₃ kinase. Horseradish peroxidase-conjugate antibodies raised in donkey against rabbit IgG (Amersham Biosciences) were used as secondary antibodies. Immunodetection was performed with an enhanced chemiluminescence kit (an ECL system, Amersham Biosciences) according to the manufacturer's directions.

	ACKNOWLEDGMENTS

 
We are sincerely grateful to Prof. Suh (Pohang University of Science and Technology, Korea) for his participation in our research collaboration. We thank Dr. Csaba Koncz (Max-Planck-Institute für Züchtungsforschung) and Dr. Masaaki Umeda (University of Tokyo) for giving Arabidopsis ecotype Columbia-0 cell suspension. We thank Kazusa DNA Research Institute for providing all expressed sequence tag clones used here. We also express our sincere appreciation to Prof. Terabe (Himeji Institute of Technology, Japan) for his kind reading and many suggestions for this manuscript. We wish to thank the Yamada Science Foundation and the Botanical Society of Japan for supporting the collaboration in Australia and Korea.

Received January 26, 2005; returned for revision February 18, 2005; accepted February 21, 2005.

	FOOTNOTES

 
¹ This work was supported by Core Research for Evolutional Science and Technology of Japan Science and Technology Agency; a Grant-in-Aid for Scientific Research on Priority Areas (B; grant no. 10219202) by the Japanese Ministry of Education, Culture, Sports, Science and Technology; a Grant-in-Aid for Scientific Research (B; grant no. 12440225) by the Japan Society for the Promotion of Science; and a Grant-in-Aid for Japan Society for the Promotion of Science Fellows by the Japan Society for the Promotion of Science. The Yamada Science Foundation, the Botanical Society of Japan, and the Australian Research Council supported the collaboration in Australia or Korea.

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	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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