JXB Advance Access published online on December 21, 2006
Journal of Experimental Botany, doi:10.1093/jxb/erl264
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RESEARCH PAPER |
Involvement of rapid nucleotide synthesis in recovery from phosphate starvation of Catharanthus roseus cells
1Department of Advanced Bioscience, Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, 112-8610 Japan
2Metabolic Biology Group, Department of Biology, Faculty of Science, Ochanomizu University, Tokyo, 112-8610 Japan
* To whom correspondence should be addressed. E-mail: ashihara.hiroshi{at}ocha.ac.jp
Received 21 September 2006; Revised 6 November 2006 Accepted 9 November 2006
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Abstract |
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Growth of suspension-cultured Catharanthus roseus cells ceased during phosphate starvation, but the cells grew again upon addition of Pi even after long-term starvation. The metabolic fate of [33P]Pi was studied in 1-week-old stationary phase cells in ordinary culture and in 1- or 2-week-old Pi-starved cells. Immediately after administration, the most heavily labelled organic compounds are nucleotides, followed by sugar phosphates. Two weeks Pi starvation slowed down the speed of incorporation of 33P into nucleotides. The RNA, protein, and free nucleotide content all decreased gradually during Pi starvation; however, these compounds, especially nucleotides, increased markedly in the 24 h after addition of Pi. These responses are found in all cells examined, although the total amounts of these compounds were lower in the long-term Pi-deficient cells. Of the nucleotides, a marked increase was observed in nucleoside triphosphates and UDP-glucose. The transcript level of phosphate transporter and the activities of acid phosphatase, 5'- and 3'-nucleotidase, and adenosine nucleosidase were all reduced by the addition of Pi. In contrast, the activities of adenine phosphoribosyltransferase, nicotinate phosphoribosyltransferase, and nicotinamidase, which are salvage enzymes of purine and pyridine nucleotides, were markedly increased in the Pi-fed cells. Little or no increase was observed in adenosine kinase. In the light of these results, the possible involvement of net nucleotide synthesis in the initial metabolic events of recovery from Pi deficiency are discussed.
Key words: Adenine nucleotide, cultured cell, metabolic regulation, nucleotide biosynthesis, phosphate starvation, salvage pathway
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Suspension-cultured Catharanthus roseus cells are useful for investigating the role of Pi on growth and metabolism. Since Amino et al. (1983) established a synchronous cell division system using Pi starvation, this culture system has been used for various Pi starvation experiments (Ashihara and Ukaji, 1986; Ukaji and Ashihara, 1987; Nagano and Ashihara, 1993; Nagano et al., 1994; Shimano and Ashihara, 2006). It has been reported previously that purine nucleotide levels decrease markedly, together with an increase in hydrolysing enzymes such as 3'- and 5'-nucleotidases and RNase, during long-tem Pi starvation (Shimano and Ashihara, 2006). These Pi-starved cells grow again when Pi is added to the culture medium. Effects of Pi starvation on carbohydrate and nucleotide metabolism have been reported (Duff et al., 1989; Theodorou et al., 1992; Nagano and Ashihara, 1993; Rychter and Randal, 1994; Murley et al., 1998; Raghothama, 1999; Stasolla et al., 2003; Shimano and Ashihara, 2006).
Transcriptomic analyses using model plants, such as Arabidopsis thaliana and rice, indicate that the expression of various genes changes when plants are grown with phosphate deficiency (Hammond et al., 2003; Uhde-Stone et al., 2003; Wu et al., 2003; Misson et al., 2005; Wasaki et al., 2006). Only a limited number of genes related to nucleotide metabolism have so far been noted. Expression of genes encoding an acid phosphatase, RNase, and Pi transporters during recovery from Pi starvation has been studied in roots and shoots of A. thaliana (Muller et al., 2004).
To determine which compound(s) are initially formed by the re-addition of Pi, and which act as triggers initiating the dramatic change in metabolism after Pi addition, the metabolic fate of exogenously supplied 33P in the Pi-deficient C. roseus cells was first monitored for 5–60 min. The earliest labelled compounds include nucleotides. Biochemical parameters related to nucleotide metabolism were therefore compared between Pi-starved and Pi-fed cells. Based on the results, a possible role in rapid nucleotide synthesis of purine and pyridine salvage enzymes is proposed during recovery from Pi starvation.
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Materials and methods |
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Chemicals
Sodium [33P]phosphate (110 GBq µmol–1) was obtained from Amersham Pharmacia Biotech, Amersham, UK.
Plant materials
Stock cultures of C. roseus (L.) G. Don were maintained at 1-week intervals in modified Murashige–Skoog basic medium (+Pi medium) containing 1.25 mM Pi, supplemented by 3% sucrose and 2.2 µM 2,4-dichlorophenoxyacetic acid (Murashige and Skoog, 1962; Linsmaier and Skoog, 1965). For preparation of experimental cultures, portions of 7-d-old cell suspension (7 ml, c.1.5 g fresh weight) were transferred to 43 ml aliquots of fresh +Pi medium in 300 ml Erlenmeyer flasks for 7 d. The resulting 7-d-old cells grown in +Pi medium (7 ml, c.1.5 g fresh weight) were transferred to 43 ml of Pi-deficient (–Pi) medium and were cultured for 2 weeks. The culture flasks were placed on a horizontal rotary shaker (90 strokes min–1, 80 mm amplitude) at 25 °C in the dark. For recovery of Pi, 0.5 ml of 125 mM Pi was added to the Pi-starved cultures under sterile conditions.
33P tracer experiments
To determine the metabolic fate of [33P]Pi, cultured cells (100 mg fresh weight) and 1.9 ml of the cultured medium in which the cells had been grown were placed in 30 ml Erlenmeyer flasks. Each reaction was started by adding 10 µl (3.7 kBq) of a solution of [33P]Pi. The flasks were incubated in an oscillating water bath at 27 °C. After incubation, the cells were harvested by filtration over Miracloth, washed with distilled water, frozen with liquid nitrogen, and stored at –80 °C. The cells were homogenized in 6% HClO4 in an ice bath using a glass homogenizer. The homogenate was extracted successively with 6% HClO4, ethanol–diethylether mixture at 50 °C for 15 min, 6% HClO4 at 100 °C for 15 min, and 3 N NaOH at 100 °C for 20 min, according to Ashihara and Tokoro (1985). The first 6% HClO4-soluble fraction contained labelled Pi, nucleotides, and sugar phosphates. The ethanol–diethylether soluble fraction, the second hot HClO4-soluble fraction, and the NaOH-soluble fraction, respectively, contain labelled phospholipids, nucleic acids, and phosphoproteins. The first HClO4-soluble fraction was neutralized with 20% KOH. After removal of potassium perchlorate by brief centrifugation, the sample was fractionated into Pi, nucleotides, and sugar phosphates by a combination of ion exchange and precipitation chromatography as established by Derr et al. (1977), using a Dowex 1-X8 (Cl– form) and Dowex 50-X8 (La3+ form) column. In some experiments, the neutralized HClO4-soluble fraction was concentrated in vacuo, and was analysed by thin-layer chromatography (TLC) as set out in a previous paper (Shimano and Ashihara, 2006). The radioactivity in each fraction was measured with a Beckman LS 6500 liquid scintillation spectrometer. Radioactivity on the TLC plates was determined using a Bio-imaging Analyser, Type FLA-2000, Fuji Photo Film Co., Ltd, Tokyo, Japan.
Analysis of RNA, protein, and free nucleotide contents
RNA and protein content was determined as in a previous report (Ukaji and Ashihara, 1986). Nucleotides were determined by the method of Ashihara et al. (1987). This procedure has been reported in detail previously (Shimano and Ashihara, 2006).
Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)
For RT–PCR, the cells were immediately frozen with liquid nitrogen. Total RNA extracted from samples was treated with RNase-free DNase I (Promega, Madison, WI, USA). DNA-free total RNA from each sample was used for first-strand cDNA synthesis. The reaction mixture (20 µl) contained 125 U of MuLV reverse transcriptase and 2.5 mM oligo d(T)16 (Applied Biosystems, Branchburg, NJ, USA). The mixture (25 µl) for the PCR contained 22.5 µl of PlatinumR PCR Supermix high Fidelity (catalogue No. 12532-016; Invitogen, Carlsbad, CA, USA), 1 µl of 10 µM of gene-specific primers, and 1.5 µl (16 ng) of cDNA. The nucleotide sequences of the primers used in this study were obtained from Kai et al. (1997) and Murata and Luca (2005).
The gene-specific oligonucleotide primers used were as follows: phosphate transporter (PIT1, AB004809 [GenBank] ) primer, 5'-TTTCCCGGCATGGTTAAGGT-3' and 5'-TGACGCAGCCTAGAACAATG-3'; actin (Cractin, no accession number provided) primer, 5'-GGCTGGATTTGCTGGAGATGAT-3' and 5'-TAGATCCTCCGATCCAGACACTG-3'.
For PCR, a Gene Amp PCR System 2400 thermal cycler was used. The amplification programmes were as follows: PIT1, 2 min at 95 °C, 25–35 cycles of 1 min at 95 °C, 1 min at 55 °C, and 1 min at 72 °C, and a final extension step of 10 min at 72 °C; Cractin: 2 min at 94 °C, 25–35 cycles of 15 s at 94 °C, 20 s at 57 °C, and 30 s at 72 °C, and a final extension step of 10 min at 72 °C The amplification showed a linear curve. The reaction product was visualized by UV light on a 2% agarose gel stained with ethidium bromide.
Preparation of enzymes
Freshly harvested C. roseus cells (
500 mg fresh weight) were homogenized with 5 ml of 50 mM TRIS–HCl buffer (pH 7.5) using a glass homogenizer, and the homogenate was centrifuged at 20 000 g for 15 min at 2 °C. The resulting supernatant fluid was brought to 80% saturation with finely ground solid ammonium sulphate and was centrifuged at 20 000 g for 15 min. To determine the activities of acid phosphatase (APase) and 5'- and 3'-nucleotidases, the ammonium sulphate precipitate was suspended in 50 mM TRIS–HCl buffer (pH 7.5) and the sample (2.5 ml) was desalted using a column of Sephadex G-25 (PD-10 column, Amersham Pharmacia Biotech Ltd, UK) that had been equilibrated with the same buffer. The eluted protein fraction (3.5 ml) was used for the assay. For RNase assay, TRIS–HCl buffer was replaced by 50 mM succinic acid–NaOH buffer (pH 5.0) containing 2.5 mM sodium EDTA. For adenosine kinase, adenine phosphoribosyltransferase (APRT), adenosine nucleosidase (AN), nicotinate phosphoribosyltransferase (NaPRT), and nicotinamidase, the relevant protein was dissolved in 50 mM HEPES–NaOH buffer (pH 7.5) containing 0.5% (w/v) sodium ascorbate and 2 mM 2-mercaptoethanol, and was then desalted.
Determination of enzyme activity
The assay methods and the composition of the reaction mixtures for APase, nucleotidases, APRT, adenosine kinase (AK), and AN are given in a previous report (Shimano and Ashihara, 2006). The reaction mixture for NaPRTs consisted of 50 mM HEPES–NaOH buffer (pH 7.6), 0.05 mM [carboxyl-14C]nicotinic acid (specific activity, 310 MBq mmol–1), 0.75 mM 5-phosphoribosyl-1-pyrophosphate (PRPP), 10 mM MgCl2, 1 mM dithiothreitol (DTT), and 1 mM ATP. The reaction mixture for nicotinamidase was 50 mM HEPES–NaOH buffer (pH 7.6) and 0.1 mM [carbonyl-14C]nicotinamide (specific activity, 170 MBq mmol–1). The total volume of the reaction mixtures was 100 µl, and incubation took place at 30 °C. The enzyme reactions were terminated by transferring the test tubes to a boiling water bath. After brief centrifugation, the precipitate was removed, and an aliquot of each sample was loaded onto the cellulose TLC plate. Labelled substrate and product were separated by TLC, using n-butanol:acetic acid:water (4:1:2, by vol.) as the solvent.
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Results |
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Growth of cells during Pi starvation and recovery
A synchronous cell division system of C. roseus was originally established using the phosphate starvation method (Amino et al., 1983). A similar, but simplified culture system was used for this study. The stationary phase (7-d-old) cells of a stock culture of C. roseus was transferred to fresh MS medium containing 1.25 mM Pi (+Pi medium) for 7 d. Figure 1 shows changes in cell numbers in Pi-free and Pi-containing MS medium. During a cycle of growth with ordinary MS medium containing Pi, cell numbers increased after cell transfer and reached the stationary phase at day 7. For Pi-deficient cells, the 7-d-old cells grown in the MS medium (‘+Pi medium’) were transferred to the fresh +Pi medium or Pi-free MS medium (‘–Pi medium’). The cells in the –Pi medium were cultured for 1 or 2 weeks. In the Pi-deficient cultures, no increase in cell numbers was observed (Fig. 1), and the cellular Pi level was maintained at a low level (Shimano and Ashihara, 2006). Cell numbers increased when Pi was added to the Pi-deficient culture (Fig. 1). It appears that C. roseus cells rest during Pi starvation, and resume growth as soon as Pi is supplied to the culture medium.
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Metabolic fate of 33P
To learn which compounds initially incorporate exogenously supplied Pi in the Pi-deficient cells, the metabolic fate of 33Pi was followed at 5, 10, 15, 30, and 60 min after addition of 33Pi (Fig. 2). Six components were fractionated using chromatography as described in an earlier report (Ashihara and Tokoro, 1985): Pi, nucleotides, sugar phosphates, phospholipids, nucleic acids, and phosphoproteins. 33Pi was immediately incorporated into the cells, and 91–100% of radioactivity was recovered in the cells. The most heavily labelled fraction in cells of day 0 and day 7 was the nucleotides; the fraction comprised 39% and 43%, respectively, of total 33P (Fig. 2A, B). In day 14 cells, 76% of Pi remained unchanged, but up to 21% of 33P was found in the nucleotide fraction (Fig. 2C). In day 0 and day 7 cells, the second 33P incorporation was observed in the free Pi fraction. Significant incorporation of 33P into the sugar phosphate fraction was also found. The fraction incorporated into nucleotides and sugar phosphates usually decreased slightly with increasing time of incubation. In contrast, incorporation into phospholipids and nucleic acids increased with time. This implies that some phosphorylated compounds were used for the synthesis of macromolecules. At 60 min after 33P addition, 6, 7, and 6% of radioactivity was distributed in nucleic acids in day 0, 7, and 14 cells, respectively (Fig. 2A–C). These results suggest that Pi was initially used for ATP synthesis from ADP by oxidative phosphorylation. Other nucleotides and sugar phosphates, such as UTP, GTP, CTP, glucose-6-phosphate, and fructose-6-phosphate, are formed via ATP by the reactions catalysed by various kinases. The degree of incorporation of 33Pi into nucleic acids is similar regardless of the duration of Pi starvation.
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Changes in contents of RNA, protein, and nucleotides
Figure 3A–C shows the levels of RNA, protein, and total free nucleotides expressed per gram fresh weight. The RNA, protein, and nucleotide content all gradually decreased with the duration of Pi starvation. At 1 d after the addition of Pi, the contents of all three increased, but the greatest changes were found in free nucleotides (Fig. 3C).
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Figure 4 gives a visual comparison of levels in individual nucleotides in Pi-starved (collected at days 0, 7, and 14) and Pi-fed cells (collected at days 1, 8, and 15). After addition of Pi, there was invariably a marked increase in contents of nucleoside triphosphates, namely ATP, GTP, UTP, and CTP, but little increment in AMP was observed. These results suggest that a key initial event in recovery from phosphate starvation is the formation of nucleoside triphosphates.
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Transcript levels of the phosphate transporter gene, PIT1
Figure 5 shows the levels of transcript of PIT1, which encodes a phosphate transporter protein in Pi-starved (days 0, 7, and 14) and Pi-fed (days 1, 8, and 15) C. roseus cells.
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The transcript levels were high during Pi starvation. After supply of Pi, the levels were reduced considerably. It is obvious in the cells at day 15.
Changes in activity of nucleotide-related degradation enzymes
As shown in a previous report (Shimano and Ashihara, 2006), activities of acid phosphatase, and 5'- and 3'-nucleotidase measured in in vitro enzyme preparations increased markedly during Pi starvation. The activities of these enzymes decreased in P-fed cells (Fig. 6A–C).
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Changes in activity of enzymes related to salvage formation of adenine nucleotides
The most efficient formation of adenine nucleotides is performed by the salvage pathways using the pool(s) of adenine and/or adenosine. The activity of APRT and AK was maintained or increased slightly in the Pi-fed cells (Fig. 7A, B). In contrast, the activity of AN, which catalyses the hydrolysis of adenosine to adenine, decreased slightly in the Pi-fed cells (Fig. 7C).
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Changes in activity of enzymes related to nicotinamide salvage
Nicotinamide, which is a degradation product of NAD, is converted to nicotinate by nicotinamidase and is salvaged by NaPRT. The activity of both enzymes decreased during Pi starvation, but increased markedly after supply of Pi (Fig. 8A, B).
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Discussion |
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Phosphorus is one of three essential macronutrients of plants (Raghothama, 1999). Several studies have recently suggested that expression of many genes depends on the Pi level in plants. For example, as is confirmed here, the phosphate transporter gene is strongly expressed in Pi-deficient plants (Dong et al., 1999; Kai et al., 2002), probably an adaptation to obtain more Pi from outside the cells. The synthesis of many mRNAs still occurs in Pi-deficient cells. However, levels of nucleotide triphosphates, i.e. substrates for RNA synthesis, are markedly decreased in the cells, and net synthesis of RNA must therefore be greatly reduced in the Pi-deficient cells. This implies that studies on the gene expression and investigation at the metabolite level are both important in determining the Pi-dependent mechanism of control of cell growth, although the latter approach is limited.
Based on the 33P feeding study, it is speculated that an initial event in recovery of Pi starvation involves the synthesis of nucleotides, especially nucleoside triphosphates. In the Pi-starved cells, AMP and ADP dominated the adenine nucleotides, and the adenylate energy charge was much lower than in the growing cells (Shimano and Ashihara, 2006). When Pi is supplied to the Pi-deficient culture, ATP is formed from ADP and Pi mainly by respiration (stage 1). Turnover, i.e. utilization and regeneration, of ATP is accelerated, and phosphate groups at the 
-position of ATP molecules are transferred to various nucleoside mono- and diphosphates and free sugars, so that many nucleoside triphosphates and sugar phosphates are formed (stage 2). The net increase in nucleotides is initiated by salvage reactions utilizing the nucleosides and nucleobases which had accumulated in the Pi-starved cells (Shimano and Ashihara, 2006). ATP and PRPP are used for nucleoside and nucleobase salvage reactions by kinase and phosphoribosyltransferase, respectively. It has been reported that the availability of PRPP in C. roseus cells is increased upon addition of Pi (Ukaji and Ashihara, 1987).
Once the total nucleotide level has increased, it triggers the net synthesis of RNA and protein (stage 3). It has been reported that free amino acids are accumulated in Pi-starved cells where the protein synthesis is limited (Ukaji and Ashihara, 1987), so that no amino acid synthesis may be needed for protein synthesis during the early phase of recovery. DNA duplication then takes place and cell division begins (stage 4). A greater increase in nucleotide level and a net increase in RNA and protein content and in cell numbers are detected in the 24 h Pi-fed cells (Figs 3, 4). A possible sequence of metabolic activation beginning from Pi starvation is shown in Fig. 9.
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The activities of hydrolysing enzymes, APase, and nucleotidases are significantly reduced in the Pi-fed cells in which the availability of free Pi increased. These findings are supported by the fact that expression of the genes of APase and some hydrolysing enzymes is rapidly reduced in Pi-fed Arabidopsis seedlings (Muller et al., 2004).
It was reported previously that activities of AK, AN, and APRT were maintained at a high level even in long-term Pi-starved cells, and served for the limited turnover of nucleotides in these cells (Shimano and Ashihara, 2006). These enzymes may also have an important function in the resumption of cell growth via the increase in net synthesis of nucleotides during the early stage of recovery from Pi starvation. Of the three enzymes measured, APRT and AK activities increased or were maintained by the addition of Pi, whereas AN was reduced in the Pi-fed cells. This differing response suggests that AN, which catalyses the hydrolysis of adenosine to adenine, is not involved in purine salvage but takes part in the degradation process.
Marked increases in the activities of nicotinamidase and NaPRT are found in the Pi-fed cells. This suggests that the turnover of NAD is accelerated in the Pi-fed cells. Salvage activity of nicotinamide, or in other words re-utilization of nicotinamide by the pyridine nucleotide cycle, was at work in the growing Pi-fed cells. In plants, in contrast to animals, no nicotinamide phosphoribosyltransferase is present, so that nicotinamide is utilized by NaPRT after hydrolysis to nicotinic acid (Ashihara et al., 2005; Zheng et al., 2005). The increase in nicotinamidase activity in the Pi-fed cells suggests that this enzyme acts as a salvage enzyme to pick up nicotinamide for NAD synthesis, but not as a simple degradation enzyme of nicotinamide.
The present results strongly suggest that rapid reformation of nucleotides by salvage reactions are key events in recovery from Pi starvation.
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Acknowledgements |
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This work was partly supported by a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science (No. 16570031).
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Abbreviations |
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AK, adenosine kinase; APase, acid phosphatase; APRT, adenine phosphoribosyltransferase; AN, adenosine nucleosidase; NaPRT, nicotinate phosphoribosyltransferase; PRPP, 5-phosphoribosyl-1-pyrophosphate RT-PCR, reverse transcription-polymerase chain reaction; TLC, thin-layer chromatography.
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References |
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