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Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices
Plant Physiology and Biochemistry 44 (2006) 551–555 www.elsevier.com/locate/plaphy
Research article
Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices Riko Katahiraa,1, Hiroshi Ashiharaa,b,* a
Department of Advanced Bioscience, Graduate School of Humanities and Sciences, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan b Metabolic Biology Group, Department of Biology, Faculty of Science, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan Received 27 April 2006; accepted 22 September 2006 Available online 09 October 2006
Abstract Levels of ATP and other nucleotides increased in wounded potato tuber slices, maintained on moist paper for 24 h after preparation. The relative expression intensity of genes encoding adenosine kinase (AK) and adenine phosphoribosyltransferase (APRT) in wounded slices was greater than the intensity of genes of the de novo pathway, glycineamide ribonucleotide formyltransferase (GART) and 5-aminoimidazole ribonucleotide synthetase (AIRS). In vitro activities of adenosine kinase (ATP:adenosine 5′-phosphotransferase; EC 2.7.1.20) and adenine phosphoribosyltransferase (AMP:pyrophosphate phospho-D-ribosyltransferase; EC 2.4.2.7) increased during wounding. Adenosine nucleosidase (adenosine ribohydrolase; EC 3.2.2.7) activity was negligible in freshly prepared slices, but its activity is dramatically enhanced in wounded slices. In situ adenosine salvage activity, estimated from the incorporation of radioactivity from exogenously supplied [8-14C]adenosine into nucleotides and RNA, increased more than five times in the wounded slices. These results strongly suggest that greater expression of the genes encoding enzymes of adenosine salvage during wounding is closely related to the increased supply of adenine nucleotides in the wounded slices. © 2006 Elsevier Masson SAS. All rights reserved. Keywords: Adenine nucleotides; Potato; Purine nucleotide metabolism; Salvage pathway; Solanum tuberosum; Wounded plant tissue
1. Introduction Activation of plant storage tissue after excision and incubation is known as the ‘wounding’ or ‘ageing’ phenomenon [9]. ‘Wound respiration’ of potato tubers has been investigated by plant physiologists since the late nineteenth century [12]. Many studies have been performed on the effect of wounding on cyanide-sensitive or cyanide-resistant respiration and mitochondrial membrane proteins [8], but there are only a few studies of the synthesis of ATP and other nucleotides during the wounding process. Takamura and Uritani [17] found that the
* Corresponding
author. E-mail address: [email protected] (H. Ashihara). 1 Permanent address: Laboratory of Microbiology, Faculty of Home Economics, Tokyo Kasei Gakuin University, Machida-shi, Tokyo 194-0292, Japan. 0981-9428/$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2006.09.016
adenine nucleotide content increased up to 1.3 times during wounding of sweet potato root tissue. Nucleotides are synthesised by the de novo pathway using amino acids and other low molecular weight compounds and by salvage pathways, which utilise bases and nucleosides derived from nucleotides and nucleic acids [1,16]. Little is known about the nucleotide metabolism in wounding potato tubers, however. In the present study, we confirm that levels of nucleotides increase markedly during wounding of potato tuber slices. To determine the mechanism of wound-induced rapid nucleotide synthesis, we first examined the transcript levels of genes encoding the enzymes of the de novo and salvage pathways of purine nucleotides. These results imply the preferential contribution of salvage pathways to enhanced biosynthesis of nucleotides in wounded slices. Enzyme activities of purine salvage pathways were therefore measured, and salvage activities in vivo were estimated from the incorporation of radioactivity from [8-14C]adenosine into nucleotides and
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nucleic acids. Based on these findings, we discuss the role of adenosine salvage in the biosynthesis of ATP in wounded potato tuber slices. This process may be closely related to the activation of plant storage tissue. 2. Results 2.1. Nucleotide content The total nucleotide content doubled during wounding of potato tuber slices, although the extent of the increase was different for the individual nucleotides. Increases by nearly three times in the levels of ATP and 1.4- to 7 times in other nucleotides were observed at 24 h after wounding (Fig. 1). The adenylate energy charge, i.e. ([ATP] + 1/2 [ADP])/([ATP] + [ADP] + [AMP]), was approximately 0.7 in fresh slices but was 0.8 in the wounded slices. The increased energy charge may be caused by the respiratory rise in wounded slices [18], but this wounding effect implies more than an increase in the conversion of ADP to ATP, because the total adenine nucleotides, including AMP and ADP, also increased up to 2.5 times in wounded slices. These results suggest that the increase in the nucleotide pool is related to the rapid and significant changes in metabolism including nucleotide biosynthesis during wounding.
GART and AIRS, which respectively, encode glycineamide ribonucleotide formyl transferase (10-formyltetrahydrofolate: 5′-phosphoribosylglycinamide N-formyltransferase; EC 2.1.2.2) and 5-aminoimidazole ribonucleotide synthetase cyclo(2-(formamido)-N1-(5-phosphoribosyl)acetamidine ligase (ADP-forming); EC 6.3.3.1), were used. These two enzymes catalyse the third and fifth steps in the de novo purine nucleotide biosynthesis. For the salvage pathway, AK and APRT genes, which code for adenosine kinase and adenine phosphoribosyltransferase, were examined. Transcript levels of all four genes were increased in wounded potato slices but, compared to the increase in the transcripts of the de novo genes, the levels of AK and APRT were greatly enhanced (Fig. 3). 2.3. Activity of enzymes involved in adenosine salvage To examine whether the increased level of transcripts of AK and APRT in the wounded slices affects the activity of these enzymes, we measured the activity of these two enzymes in
2.2. Expression of genes involved in de novo and salvage pathways The nucleotides are synthesised by both the de novo and salvage pathways [16]. Fig. 2 outlines the de novo and salvage pathways leading to ATP synthesis. We chose four genes to investigate the expression of genes encoding enzymes of the de novo and the salvage pathways. For the de novo pathway,
Fig. 1. Comparison of nucleotide content in fresh and wounded slices of potato tubers. Nucleotide content is expressed as nmol g–1 fresh weight. Mean values and S.D. (N = 3) are shown. Values for wounded slices marked with asterisks differ significantly from the values for fresh slices according to the t-test (P < 0.05).
Fig. 2. Metabolic pathways for ATP biosynthesis. The de novo and salvage pathways of adenine nucleotide synthesis are shown. Expression of genes shown in italics and activity of enzymes in numbers were studied in this study. (1) Adenosine kinase, (2) adenosine nucleosidase, and (3) adenine phosphoribosyltransferase. AIR, 5-aminoimidazole ribonucleotide; FGAM, formylglycine amidine ribonucleotide; FGAR, formylglycineamide ribonucleotide; GAR, glycineamide ribonucleotide; PRPP, 5-phosphoribosyl-1-pyrophosphate; THF, tetrahydrofolate.
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Fig. 3. Relative transcript levels of GART, AIRS, AK and APRT in fresh and wounded slices of potato tubers. The transcript levels were analysed by quantitative real-time PCR and were normalised to those of the freshly prepared slices. Relative rates are shown as % of the values observed in fresh slices, with S.D. (N = 3).
extracts from fresh and wounded tuber slices. The activity of adenosine nucleosidase, which is also involved in adenosine salvage, was determined (Fig. 4). The gene for the nucleosidase has not yet been cloned, so we could not test for gene expression. Activities of adenosine kinase and adenine phosphoribosyltransferase were doubled in the wounded slices, and a more profound development of adenosine nucleosidase activity was observed during wounding. Adenosine may therefore be more readily converted to adenine and salvaged by adenine phosphoribosyltransferase in wounded slices. 2.4. In situ synthesis of adenine nucleotides from [8-14C]adenosine The rate of salvage synthesis of adenine nucleotides in vivo was estimated from the incorporation of radioactivity from
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Fig. 5. In situ biosynthesis of adenine nucleotides and nucleic acids from [8-14C]adenosine in fresh and wounded potato tuber slices. Mean biosynthetic activities are shown, expressed as nmol g–1 fresh weight per 2 hours, and S.D. (N = 3). Values for wounded slices marked with asterisks differ significantly from the value for fresh slices according to the t-test (P < 0.05).
[8-14C]adenosine into adenine nucleotides and nucleic acids for 2 h (Fig. 5). The rates of incorporation into adenine nucleotides and into nucleic acids in the wounded slices was, respectively, 5.8- and 4.0-times higher than in fresh slices. Essentially all [8-14C]adenosine taken up by the freshly prepared and wounded slices was converted to the salvage compounds. Less than 4% of radioactivity was found in degradation products, mainly ureides, in both slices. 3. Discussion The remarkable increase in adenosine and adenine salvage activities in wounded tubers may participate in the salvage of adenine and/or adenosine, which are produced by rapid turnover of ATP in wounded potato slices. These salvage pathways may also contribute to the recycling of nucleosides derived from RNA. Sacher et al. [14] observed a fourfold increase in activity of RNases at 24 h after wounding of potato tubers. The net RNA content of wounded slices (0.98 mg g–1 fresh weight) was higher than in fresh slices (0.80 mg g–1 fresh weight), so
Fig. 4. Activities of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase in fresh and wounded potato tuber slices. Values for wounded slices marked with asterisks differ significantly from the value for fresh slices according to the t-test (P < 0.05).
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that the turnover of labile RNA, such as mRNA, may also increase during wounding. There are two possible ways to increase the net nucleotide contents: one is nucleotide synthesis from amino acids and small molecular compounds by the de novo pathway, the other is synthesis from nucleosides and/or nucleobases derived from RNA stored in dormant potato tubers. An increase in the latter process has been observed in germinating Phaseolus mungo seeds [2]. However, in potato tubers, no drastic decrease in RNA content was found during wounding, so that this possibility seems implausible. Although there is some evidence that dormancy breaking of plant buds and tubers is induced by the enhanced activity of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase [7,11,13,16], no data exists on the expression of genes encoding for these enzymes. From an energetics viewpoint, the wounding process of potato tuber slices in this study is similar to the dormancy break of intact potato tubers. Accelerated energy generation is required for these processes. In order to maintain high levels of ATP in potato tuber slices after wounding, the salvage pathways of adenine and adenosine seem to be very effective. The present results suggest that the enhanced adenine and adenosine salvage activity in the wounded tuber slices is directly attributable to the pronounced expression of AK and APRT, and the subsequent increase in AK and APRT activities. 4. Materials and methods 4.1. Plant materials Mature tubers of potato (Solanum tuberosum L., cv. Danshaku) were obtained from Imagane-cho, Hokkaido, Japan. The potato tubers were ‘wounded’ according to the method of Dyer et al. [6]. In summary, cylinders (diameter 7 mm) were cut from tubers with a sterilised cork borer, the epidermis was removed, and the cores were sliced into 1.5-mm-thick sections with a razor blade. All tissues were maintained on moist sterile filter paper in Petri dishes at 25 °C for 24 h. Freshly cut and wounded slices were frozen in liquid nitrogen for the determination of nucleotides and RNA isolation. 4.2. Determination of nucleotide content Frozen slices (500 mg fresh weight) were crushed in liquid nitrogen with a mortar and pestle, and extracted with precooled 18% (w/v) trichloroacetic acid including 5 mM NaEGTA for 2 h at 0 °C. Trichloroacetic acid in the extracts was removed with water-saturated diethyl ether, and the extracts were neutralised with 1 M triethanolamine containing 5 M KOH. The neutralised extracts were freeze-dried and redissolved in 20 mM KH2PO4–NaH2PO4 buffer (pH 7.0). The nucleotide content was determined by HPLC using an anionexchange Shimpack WAX-1 column (Shimadzu Corp., Kyoto, Japan) according to the method by Ashihara et al. [3]. The original time program of the gradient of (A) 20 mM
KH2PO4–NaH2PO4 (pH 7.0) and (B) 480 mM KH2PO4–NaH2PO4 (pH 6.85) was slightly modified to improve the separation of GDP and UTP: 0–22 min, 0–50% B; 22–30 min, 50–60% B; 30–35 min, 60–100% B. 4.3. Quantitative reverse transcription (RT)-PCR For real time RT-PCR, total RNA was isolated from slices (2 g fresh weight) using phenol/sodium dodecyl sulphate and precipitation with lithium chloride [15], and was treated with RNase-free DNase I (Promega, Madison, WI, USA). cDNA was prepared from 0.4 μg total RNA using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA, USA) in a reaction volume of 20 μl. Quantitative real-time PCR reactions were performed in triplicate for each cDNA reaction using 1 μl of each reverse transcribed sample, and 0.2 μM of each primer in a 10 μl 1 x formulation of Syber Green Master Mix (Applied Biosystems). The gene-specific primers used were as follows: ● GART (AY424958), 5′-GAGGAGGATCTCGTGGGTTC-3′ and 5′-GTAGCCTTTGCCTCCAAAAG-3′; ● AIRS (AY429421), 5′-ATCATGTGTACCCAAGCTCC-3′ and 5′-AGGTGCCATTTTGGCTATTC-3′; ● AK (DQ268864), 5′-CAGAGTGGCTCAGTGGATGC-3′ and 5′-TCGCCATCTAGCACACAAAC-3′; ● APRT (DQ284483), 5′-TCCCATTGCATTGGCTATTG-3′ and 5′-TCAACTCCAACACGCTCAAG-3′. The amplicons corresponding to GART, AIRS, AK and APRT were, respectively, 159, 226, 180 and 228 bp in length. An ABI Prism 7300 Sequence Detection System (Applied Biosystems) was used for thermal cycling. The PCR reactions were initiated with denaturation at 95 °C for 10 min. The cycling protocol consisted of: denaturation at 95 °C for 15 s, annealing at 57 °C for 30 s, and extension at 72 °C for 40 s, repeated for up to 40 cycles. 4.4. Determination of enzyme activity Enzyme extraction was performed exactly as described in our previous paper [10]. Potato tuber slices (ca. 2 g, fresh weight) were homogenised in a chilled extraction medium containing 50 mM Hepes-NaOH buffer (pH 7.6), 2 mM NaEDTA, 2 mM dithiothreitol, 0.5% sodium ascorbate and 2% polyvinylpolypyrrolidone, using a pestle and mortar on ice. The homogenate was centrifuged at 20,000 × g for 20 min at 4 °C. Aliquots of the supernatant (2.5 ml) were desalted on a pre-packed column of Sephadex G-25 (PD-10) (Amersham Pharmacia Biotech, Uppsala, Sweden), and were used for enzyme preparation. The soluble protein content was determined using the dye-binding assay method [5] with bovine serum albumin as standard. The activity of enzymes was determined radiochemically according to the method described in [4]. The specific activities of the labelled substrates used for the radiochemical enzyme assays were adjusted to 340 kBq μmol–1 using non-
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radioactive compounds. The reaction mixtures for the enzyme assay contained 30 mM Hepes-NaOH buffer (pH 7.6), 10 mM MgCl2, 1 mM dithiothreitol, and substrates as follows: adenine phosphoribosyltransferase, 0.6 mM 5-phosphoribosyl-1-pyrophosphate and 55 μM [8-14C]adenine; adenosine kinase, 3.75 mM ATP and 55 μM [8-14C]adenosine; and adenosine nucleosidase, 55 μM [8-14C]adenosine. For determination of adenosine kinase activity, an ATP generating system was used consisting of 1 mM phosphoenolpyruvate and 16.7 nkat rabbit muscle pyruvate kinase (Sigma No. P9136); a phosphatase inhibitor (10 mM NaF) was added to the reaction mixture to prevent decomposition of ATP by non-specific phosphatases, which were present in the enzyme preparation. The reaction mixture (total volume 100 μl) was incubated at 30 °C, and the reaction was terminated by adding 10 μl of 60% perchloric acid. Denatured protein was removed by brief centrifuging. The supernatant was then neutralised with 20% KOH. After removal of potassium perchlorate by centrifuging, the supernatant was evaporated to dryness. The pellets were dissolved in 50% (v/v) ethanol and loaded onto cellulose TLC plates (Merck, Darmstadt, Germany). The labelled substrate and product were separated by TLC with two solvent systems: (I) n-butanol/acetic acid/water (4:1:2, v/v/v) and (II) distilled water, as described in our previous paper [10]. Proportionality between the reaction velocity and the amount of enzyme was confirmed by plotting the initial velocities against at least three different amounts of the enzyme preparation. 4.5. Determination of adenosine salvage in potato slices In situ metabolism of [8-14C]adenosine in freshly prepared and wounded slices was investigated essentially as in our previous paper [10]. Tuber slices (ca. 400 mg fresh weight) and 1.8 ml of sterilised 20 mM potassium phosphate buffer (pH 5.7) were placed in the main compartment of a 30-ml Erlenmeyer flask fitted with a glass tube containing a piece of filter paper impregnated with 0.1 ml of 20% KOH in a centre well. The tracer experiments were started by adding 10 μl (37 kBq) of [8-14C]adenosine (specific activity, 2.04 GBq mmol–1), and samples were incubated in an oscillating water bath at 25 °C for 2 h. After incubation the slices were collected, washed with distilled water, frozen with liquid nitrogen and then stored at –80 °C. Simultaneously, the glass tube was removed from the centre well and placed in a 50-ml Erlenmeyer flask containing 10 ml of distilled water. Potassium bicarbonate that had been absorbed by the filter paper was allowed to diffuse into distilled water overnight, and aliquots of the resulting solution were used for determination of the radioactivity. Slices were extracted successively with cold 6% perchloric acid, a mixture of ethanol and diethylether (1:1, v/v) at 50 °C for 15 min, and 6% perchloric acid at 100 °C for 15 min. The perchloric acidsoluble fraction was neutralised and concentrated, and labelled metabolites were separated by TLC as shown above. Nucleic acids (DNA plus RNA) were hydrolysed with 6% perchloric acid at 100 °C for 15 min. Radioactivity in the nucleic acid fraction was found only in adenine.
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Radioactivity in liquid samples was determined using a multi-purpose scintillation counter (Beckman, Type LS 6500, Fullerton, CA, USA) with scintillation fluid ACS-II (Amersham Pharmacia Biotech). The distribution of radioactivity of 14 C on the TLC plates was examined using a bio-imaging analyser (Type FLA-2000, Fuji Photo Film Co. Ltd., Tokyo, Japan). Student’s t-test was applied to determine statistical differences between values of freshly prepared and wounded slices, using the algorithm integrated into Microsoft Excel 2003 (Microsoft, Seattle). References [1] P.B. Adams, K.S. Rowan, Glycolytic control of respiration during aging of carrot root tissue, Plant Physiol. 45 (1970) 490–494. [2] H. Ashihara, Changes in activities of purine salvage and ureide synthesis during germination of black gram (Phaseolus mungo) seeds, Z. Pflanzenphysiol. 113 (1983) 47–60. [3] H. Ashihara, K. Mitsui, T. Ukaji, A simple analysis of purine and pyrimidine nucleotides in plant cells by high-performance liquid chromatography, Z. Naturforsch. 42c (1987) 290–297. [4] H. Ashihara, C. Stasolla, N. Loukanina, T.A. Thorpe, Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: metabolic fate of 14C-labeled precursors and activity of key enzymes, Physiol. Plant. 108 (2000) 25–33. [5] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [6] W.E. Dyer, J.M. Henstrand, A.K. Handa, K.M. Herrmann, Wounding induces the first enzyme of the shikimate pathway in Solanaceae, Proc. Natl. Acad. Sci. USA 86 (1989) 7370–7373. [7] F. Faye, F. Le Floc’h, Adenosine kinase of peach tree flower buds: purification and properties, Plant Physiol. Biochem. 35 (1997) 15–22. [8] C. Hiser, L. McIntosh, Alternative oxidase of potato is an integral membrane protein synthesized de novo during aging of tuber slices, Plant Physiol. 93 (1990) 312–318. [9] G. Kahl, Metabolism of plant storage tissue slices, Bot. Rev. 40 (1974) 263–314. [10] R. Katahira, H. Ashihara, Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers, Planta 215 (2002) 821–828. [11] F. Le Floc’h, J. Lafleuriel, Évolution des activités enzymatiques des voies de recyclage des bases et nucléosides puriques dans les tubercules de Topinambour, au cours de la levée de la dormance, C. R. Acad. Sc. Paris 298 (1984) 69–72. [12] H. Muller-Thurgau, Über Zuckeranhaüfung in Pflanzentheilen in Folge niederer Temperatur, Landw. Jahrb. 5 (1882) 751–828. [13] F. Robert, G. Petel, Nucleotide synthesis capability as a marker of the petiole elongation of strawberry plants (Fragaria × ananassa Duch.), J. Hortic. Sci. Biotechnol. 75 (2001) 690–696. [14] J. Sacher, G. Towers, D.D. Davies, Effect of light and ageing on enzymes, particularly phenylalanine ammonia lyase, in discs of storage tissue, Phytochemistry 11 (1972) 2383–2391. [15] M. Shirzadegan, P. Christie, J.R. Seemann, An efficient method for isolation of RNA from tissue cultured plant cells, Nucleic Acids Res. 19 (1991) 6055. [16] C. Stasolla, R. Katahira, T.A. Thorpe, H. Ashihara, Purine and pyrimidine nucleotide metabolism in higher plants, J. Plant Physiol. 160 (2003) 1271–1295. [17] T. Takamura, I. Uritani, Changes in acid-soluble nucleotides in cutinjured sweet potato root tissue, Agric. Biol. Chem. 37 (1973) 1511– 1515. [18] M. Teramoto, C. Koshiishi, H. Ashihara, Wound-induced respiration and pyrophosphate: fructose-6-phosphate phosphotransferase in potato tubers, Z. Naturforsch. 55c (2000) 953–956.
Research article
Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices Riko Katahiraa,1, Hiroshi Ashiharaa,b,* a
Department of Advanced Bioscience, Graduate School of Humanities and Sciences, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan b Metabolic Biology Group, Department of Biology, Faculty of Science, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan Received 27 April 2006; accepted 22 September 2006 Available online 09 October 2006
Abstract Levels of ATP and other nucleotides increased in wounded potato tuber slices, maintained on moist paper for 24 h after preparation. The relative expression intensity of genes encoding adenosine kinase (AK) and adenine phosphoribosyltransferase (APRT) in wounded slices was greater than the intensity of genes of the de novo pathway, glycineamide ribonucleotide formyltransferase (GART) and 5-aminoimidazole ribonucleotide synthetase (AIRS). In vitro activities of adenosine kinase (ATP:adenosine 5′-phosphotransferase; EC 2.7.1.20) and adenine phosphoribosyltransferase (AMP:pyrophosphate phospho-D-ribosyltransferase; EC 2.4.2.7) increased during wounding. Adenosine nucleosidase (adenosine ribohydrolase; EC 3.2.2.7) activity was negligible in freshly prepared slices, but its activity is dramatically enhanced in wounded slices. In situ adenosine salvage activity, estimated from the incorporation of radioactivity from exogenously supplied [8-14C]adenosine into nucleotides and RNA, increased more than five times in the wounded slices. These results strongly suggest that greater expression of the genes encoding enzymes of adenosine salvage during wounding is closely related to the increased supply of adenine nucleotides in the wounded slices. © 2006 Elsevier Masson SAS. All rights reserved. Keywords: Adenine nucleotides; Potato; Purine nucleotide metabolism; Salvage pathway; Solanum tuberosum; Wounded plant tissue
1. Introduction Activation of plant storage tissue after excision and incubation is known as the ‘wounding’ or ‘ageing’ phenomenon [9]. ‘Wound respiration’ of potato tubers has been investigated by plant physiologists since the late nineteenth century [12]. Many studies have been performed on the effect of wounding on cyanide-sensitive or cyanide-resistant respiration and mitochondrial membrane proteins [8], but there are only a few studies of the synthesis of ATP and other nucleotides during the wounding process. Takamura and Uritani [17] found that the
* Corresponding
author. E-mail address: [email protected] (H. Ashihara). 1 Permanent address: Laboratory of Microbiology, Faculty of Home Economics, Tokyo Kasei Gakuin University, Machida-shi, Tokyo 194-0292, Japan. 0981-9428/$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2006.09.016
adenine nucleotide content increased up to 1.3 times during wounding of sweet potato root tissue. Nucleotides are synthesised by the de novo pathway using amino acids and other low molecular weight compounds and by salvage pathways, which utilise bases and nucleosides derived from nucleotides and nucleic acids [1,16]. Little is known about the nucleotide metabolism in wounding potato tubers, however. In the present study, we confirm that levels of nucleotides increase markedly during wounding of potato tuber slices. To determine the mechanism of wound-induced rapid nucleotide synthesis, we first examined the transcript levels of genes encoding the enzymes of the de novo and salvage pathways of purine nucleotides. These results imply the preferential contribution of salvage pathways to enhanced biosynthesis of nucleotides in wounded slices. Enzyme activities of purine salvage pathways were therefore measured, and salvage activities in vivo were estimated from the incorporation of radioactivity from [8-14C]adenosine into nucleotides and
552
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nucleic acids. Based on these findings, we discuss the role of adenosine salvage in the biosynthesis of ATP in wounded potato tuber slices. This process may be closely related to the activation of plant storage tissue. 2. Results 2.1. Nucleotide content The total nucleotide content doubled during wounding of potato tuber slices, although the extent of the increase was different for the individual nucleotides. Increases by nearly three times in the levels of ATP and 1.4- to 7 times in other nucleotides were observed at 24 h after wounding (Fig. 1). The adenylate energy charge, i.e. ([ATP] + 1/2 [ADP])/([ATP] + [ADP] + [AMP]), was approximately 0.7 in fresh slices but was 0.8 in the wounded slices. The increased energy charge may be caused by the respiratory rise in wounded slices [18], but this wounding effect implies more than an increase in the conversion of ADP to ATP, because the total adenine nucleotides, including AMP and ADP, also increased up to 2.5 times in wounded slices. These results suggest that the increase in the nucleotide pool is related to the rapid and significant changes in metabolism including nucleotide biosynthesis during wounding.
GART and AIRS, which respectively, encode glycineamide ribonucleotide formyl transferase (10-formyltetrahydrofolate: 5′-phosphoribosylglycinamide N-formyltransferase; EC 2.1.2.2) and 5-aminoimidazole ribonucleotide synthetase cyclo(2-(formamido)-N1-(5-phosphoribosyl)acetamidine ligase (ADP-forming); EC 6.3.3.1), were used. These two enzymes catalyse the third and fifth steps in the de novo purine nucleotide biosynthesis. For the salvage pathway, AK and APRT genes, which code for adenosine kinase and adenine phosphoribosyltransferase, were examined. Transcript levels of all four genes were increased in wounded potato slices but, compared to the increase in the transcripts of the de novo genes, the levels of AK and APRT were greatly enhanced (Fig. 3). 2.3. Activity of enzymes involved in adenosine salvage To examine whether the increased level of transcripts of AK and APRT in the wounded slices affects the activity of these enzymes, we measured the activity of these two enzymes in
2.2. Expression of genes involved in de novo and salvage pathways The nucleotides are synthesised by both the de novo and salvage pathways [16]. Fig. 2 outlines the de novo and salvage pathways leading to ATP synthesis. We chose four genes to investigate the expression of genes encoding enzymes of the de novo and the salvage pathways. For the de novo pathway,
Fig. 1. Comparison of nucleotide content in fresh and wounded slices of potato tubers. Nucleotide content is expressed as nmol g–1 fresh weight. Mean values and S.D. (N = 3) are shown. Values for wounded slices marked with asterisks differ significantly from the values for fresh slices according to the t-test (P < 0.05).
Fig. 2. Metabolic pathways for ATP biosynthesis. The de novo and salvage pathways of adenine nucleotide synthesis are shown. Expression of genes shown in italics and activity of enzymes in numbers were studied in this study. (1) Adenosine kinase, (2) adenosine nucleosidase, and (3) adenine phosphoribosyltransferase. AIR, 5-aminoimidazole ribonucleotide; FGAM, formylglycine amidine ribonucleotide; FGAR, formylglycineamide ribonucleotide; GAR, glycineamide ribonucleotide; PRPP, 5-phosphoribosyl-1-pyrophosphate; THF, tetrahydrofolate.
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Fig. 3. Relative transcript levels of GART, AIRS, AK and APRT in fresh and wounded slices of potato tubers. The transcript levels were analysed by quantitative real-time PCR and were normalised to those of the freshly prepared slices. Relative rates are shown as % of the values observed in fresh slices, with S.D. (N = 3).
extracts from fresh and wounded tuber slices. The activity of adenosine nucleosidase, which is also involved in adenosine salvage, was determined (Fig. 4). The gene for the nucleosidase has not yet been cloned, so we could not test for gene expression. Activities of adenosine kinase and adenine phosphoribosyltransferase were doubled in the wounded slices, and a more profound development of adenosine nucleosidase activity was observed during wounding. Adenosine may therefore be more readily converted to adenine and salvaged by adenine phosphoribosyltransferase in wounded slices. 2.4. In situ synthesis of adenine nucleotides from [8-14C]adenosine The rate of salvage synthesis of adenine nucleotides in vivo was estimated from the incorporation of radioactivity from
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Fig. 5. In situ biosynthesis of adenine nucleotides and nucleic acids from [8-14C]adenosine in fresh and wounded potato tuber slices. Mean biosynthetic activities are shown, expressed as nmol g–1 fresh weight per 2 hours, and S.D. (N = 3). Values for wounded slices marked with asterisks differ significantly from the value for fresh slices according to the t-test (P < 0.05).
[8-14C]adenosine into adenine nucleotides and nucleic acids for 2 h (Fig. 5). The rates of incorporation into adenine nucleotides and into nucleic acids in the wounded slices was, respectively, 5.8- and 4.0-times higher than in fresh slices. Essentially all [8-14C]adenosine taken up by the freshly prepared and wounded slices was converted to the salvage compounds. Less than 4% of radioactivity was found in degradation products, mainly ureides, in both slices. 3. Discussion The remarkable increase in adenosine and adenine salvage activities in wounded tubers may participate in the salvage of adenine and/or adenosine, which are produced by rapid turnover of ATP in wounded potato slices. These salvage pathways may also contribute to the recycling of nucleosides derived from RNA. Sacher et al. [14] observed a fourfold increase in activity of RNases at 24 h after wounding of potato tubers. The net RNA content of wounded slices (0.98 mg g–1 fresh weight) was higher than in fresh slices (0.80 mg g–1 fresh weight), so
Fig. 4. Activities of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase in fresh and wounded potato tuber slices. Values for wounded slices marked with asterisks differ significantly from the value for fresh slices according to the t-test (P < 0.05).
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that the turnover of labile RNA, such as mRNA, may also increase during wounding. There are two possible ways to increase the net nucleotide contents: one is nucleotide synthesis from amino acids and small molecular compounds by the de novo pathway, the other is synthesis from nucleosides and/or nucleobases derived from RNA stored in dormant potato tubers. An increase in the latter process has been observed in germinating Phaseolus mungo seeds [2]. However, in potato tubers, no drastic decrease in RNA content was found during wounding, so that this possibility seems implausible. Although there is some evidence that dormancy breaking of plant buds and tubers is induced by the enhanced activity of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase [7,11,13,16], no data exists on the expression of genes encoding for these enzymes. From an energetics viewpoint, the wounding process of potato tuber slices in this study is similar to the dormancy break of intact potato tubers. Accelerated energy generation is required for these processes. In order to maintain high levels of ATP in potato tuber slices after wounding, the salvage pathways of adenine and adenosine seem to be very effective. The present results suggest that the enhanced adenine and adenosine salvage activity in the wounded tuber slices is directly attributable to the pronounced expression of AK and APRT, and the subsequent increase in AK and APRT activities. 4. Materials and methods 4.1. Plant materials Mature tubers of potato (Solanum tuberosum L., cv. Danshaku) were obtained from Imagane-cho, Hokkaido, Japan. The potato tubers were ‘wounded’ according to the method of Dyer et al. [6]. In summary, cylinders (diameter 7 mm) were cut from tubers with a sterilised cork borer, the epidermis was removed, and the cores were sliced into 1.5-mm-thick sections with a razor blade. All tissues were maintained on moist sterile filter paper in Petri dishes at 25 °C for 24 h. Freshly cut and wounded slices were frozen in liquid nitrogen for the determination of nucleotides and RNA isolation. 4.2. Determination of nucleotide content Frozen slices (500 mg fresh weight) were crushed in liquid nitrogen with a mortar and pestle, and extracted with precooled 18% (w/v) trichloroacetic acid including 5 mM NaEGTA for 2 h at 0 °C. Trichloroacetic acid in the extracts was removed with water-saturated diethyl ether, and the extracts were neutralised with 1 M triethanolamine containing 5 M KOH. The neutralised extracts were freeze-dried and redissolved in 20 mM KH2PO4–NaH2PO4 buffer (pH 7.0). The nucleotide content was determined by HPLC using an anionexchange Shimpack WAX-1 column (Shimadzu Corp., Kyoto, Japan) according to the method by Ashihara et al. [3]. The original time program of the gradient of (A) 20 mM
KH2PO4–NaH2PO4 (pH 7.0) and (B) 480 mM KH2PO4–NaH2PO4 (pH 6.85) was slightly modified to improve the separation of GDP and UTP: 0–22 min, 0–50% B; 22–30 min, 50–60% B; 30–35 min, 60–100% B. 4.3. Quantitative reverse transcription (RT)-PCR For real time RT-PCR, total RNA was isolated from slices (2 g fresh weight) using phenol/sodium dodecyl sulphate and precipitation with lithium chloride [15], and was treated with RNase-free DNase I (Promega, Madison, WI, USA). cDNA was prepared from 0.4 μg total RNA using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA, USA) in a reaction volume of 20 μl. Quantitative real-time PCR reactions were performed in triplicate for each cDNA reaction using 1 μl of each reverse transcribed sample, and 0.2 μM of each primer in a 10 μl 1 x formulation of Syber Green Master Mix (Applied Biosystems). The gene-specific primers used were as follows: ● GART (AY424958), 5′-GAGGAGGATCTCGTGGGTTC-3′ and 5′-GTAGCCTTTGCCTCCAAAAG-3′; ● AIRS (AY429421), 5′-ATCATGTGTACCCAAGCTCC-3′ and 5′-AGGTGCCATTTTGGCTATTC-3′; ● AK (DQ268864), 5′-CAGAGTGGCTCAGTGGATGC-3′ and 5′-TCGCCATCTAGCACACAAAC-3′; ● APRT (DQ284483), 5′-TCCCATTGCATTGGCTATTG-3′ and 5′-TCAACTCCAACACGCTCAAG-3′. The amplicons corresponding to GART, AIRS, AK and APRT were, respectively, 159, 226, 180 and 228 bp in length. An ABI Prism 7300 Sequence Detection System (Applied Biosystems) was used for thermal cycling. The PCR reactions were initiated with denaturation at 95 °C for 10 min. The cycling protocol consisted of: denaturation at 95 °C for 15 s, annealing at 57 °C for 30 s, and extension at 72 °C for 40 s, repeated for up to 40 cycles. 4.4. Determination of enzyme activity Enzyme extraction was performed exactly as described in our previous paper [10]. Potato tuber slices (ca. 2 g, fresh weight) were homogenised in a chilled extraction medium containing 50 mM Hepes-NaOH buffer (pH 7.6), 2 mM NaEDTA, 2 mM dithiothreitol, 0.5% sodium ascorbate and 2% polyvinylpolypyrrolidone, using a pestle and mortar on ice. The homogenate was centrifuged at 20,000 × g for 20 min at 4 °C. Aliquots of the supernatant (2.5 ml) were desalted on a pre-packed column of Sephadex G-25 (PD-10) (Amersham Pharmacia Biotech, Uppsala, Sweden), and were used for enzyme preparation. The soluble protein content was determined using the dye-binding assay method [5] with bovine serum albumin as standard. The activity of enzymes was determined radiochemically according to the method described in [4]. The specific activities of the labelled substrates used for the radiochemical enzyme assays were adjusted to 340 kBq μmol–1 using non-
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radioactive compounds. The reaction mixtures for the enzyme assay contained 30 mM Hepes-NaOH buffer (pH 7.6), 10 mM MgCl2, 1 mM dithiothreitol, and substrates as follows: adenine phosphoribosyltransferase, 0.6 mM 5-phosphoribosyl-1-pyrophosphate and 55 μM [8-14C]adenine; adenosine kinase, 3.75 mM ATP and 55 μM [8-14C]adenosine; and adenosine nucleosidase, 55 μM [8-14C]adenosine. For determination of adenosine kinase activity, an ATP generating system was used consisting of 1 mM phosphoenolpyruvate and 16.7 nkat rabbit muscle pyruvate kinase (Sigma No. P9136); a phosphatase inhibitor (10 mM NaF) was added to the reaction mixture to prevent decomposition of ATP by non-specific phosphatases, which were present in the enzyme preparation. The reaction mixture (total volume 100 μl) was incubated at 30 °C, and the reaction was terminated by adding 10 μl of 60% perchloric acid. Denatured protein was removed by brief centrifuging. The supernatant was then neutralised with 20% KOH. After removal of potassium perchlorate by centrifuging, the supernatant was evaporated to dryness. The pellets were dissolved in 50% (v/v) ethanol and loaded onto cellulose TLC plates (Merck, Darmstadt, Germany). The labelled substrate and product were separated by TLC with two solvent systems: (I) n-butanol/acetic acid/water (4:1:2, v/v/v) and (II) distilled water, as described in our previous paper [10]. Proportionality between the reaction velocity and the amount of enzyme was confirmed by plotting the initial velocities against at least three different amounts of the enzyme preparation. 4.5. Determination of adenosine salvage in potato slices In situ metabolism of [8-14C]adenosine in freshly prepared and wounded slices was investigated essentially as in our previous paper [10]. Tuber slices (ca. 400 mg fresh weight) and 1.8 ml of sterilised 20 mM potassium phosphate buffer (pH 5.7) were placed in the main compartment of a 30-ml Erlenmeyer flask fitted with a glass tube containing a piece of filter paper impregnated with 0.1 ml of 20% KOH in a centre well. The tracer experiments were started by adding 10 μl (37 kBq) of [8-14C]adenosine (specific activity, 2.04 GBq mmol–1), and samples were incubated in an oscillating water bath at 25 °C for 2 h. After incubation the slices were collected, washed with distilled water, frozen with liquid nitrogen and then stored at –80 °C. Simultaneously, the glass tube was removed from the centre well and placed in a 50-ml Erlenmeyer flask containing 10 ml of distilled water. Potassium bicarbonate that had been absorbed by the filter paper was allowed to diffuse into distilled water overnight, and aliquots of the resulting solution were used for determination of the radioactivity. Slices were extracted successively with cold 6% perchloric acid, a mixture of ethanol and diethylether (1:1, v/v) at 50 °C for 15 min, and 6% perchloric acid at 100 °C for 15 min. The perchloric acidsoluble fraction was neutralised and concentrated, and labelled metabolites were separated by TLC as shown above. Nucleic acids (DNA plus RNA) were hydrolysed with 6% perchloric acid at 100 °C for 15 min. Radioactivity in the nucleic acid fraction was found only in adenine.
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Radioactivity in liquid samples was determined using a multi-purpose scintillation counter (Beckman, Type LS 6500, Fullerton, CA, USA) with scintillation fluid ACS-II (Amersham Pharmacia Biotech). The distribution of radioactivity of 14 C on the TLC plates was examined using a bio-imaging analyser (Type FLA-2000, Fuji Photo Film Co. Ltd., Tokyo, Japan). Student’s t-test was applied to determine statistical differences between values of freshly prepared and wounded slices, using the algorithm integrated into Microsoft Excel 2003 (Microsoft, Seattle). References [1] P.B. Adams, K.S. Rowan, Glycolytic control of respiration during aging of carrot root tissue, Plant Physiol. 45 (1970) 490–494. [2] H. Ashihara, Changes in activities of purine salvage and ureide synthesis during germination of black gram (Phaseolus mungo) seeds, Z. Pflanzenphysiol. 113 (1983) 47–60. [3] H. Ashihara, K. Mitsui, T. Ukaji, A simple analysis of purine and pyrimidine nucleotides in plant cells by high-performance liquid chromatography, Z. Naturforsch. 42c (1987) 290–297. [4] H. Ashihara, C. Stasolla, N. Loukanina, T.A. Thorpe, Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: metabolic fate of 14C-labeled precursors and activity of key enzymes, Physiol. Plant. 108 (2000) 25–33. [5] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [6] W.E. Dyer, J.M. Henstrand, A.K. Handa, K.M. Herrmann, Wounding induces the first enzyme of the shikimate pathway in Solanaceae, Proc. Natl. Acad. Sci. USA 86 (1989) 7370–7373. [7] F. Faye, F. Le Floc’h, Adenosine kinase of peach tree flower buds: purification and properties, Plant Physiol. Biochem. 35 (1997) 15–22. [8] C. Hiser, L. McIntosh, Alternative oxidase of potato is an integral membrane protein synthesized de novo during aging of tuber slices, Plant Physiol. 93 (1990) 312–318. [9] G. Kahl, Metabolism of plant storage tissue slices, Bot. Rev. 40 (1974) 263–314. [10] R. Katahira, H. Ashihara, Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers, Planta 215 (2002) 821–828. [11] F. Le Floc’h, J. Lafleuriel, Évolution des activités enzymatiques des voies de recyclage des bases et nucléosides puriques dans les tubercules de Topinambour, au cours de la levée de la dormance, C. R. Acad. Sc. Paris 298 (1984) 69–72. [12] H. Muller-Thurgau, Über Zuckeranhaüfung in Pflanzentheilen in Folge niederer Temperatur, Landw. Jahrb. 5 (1882) 751–828. [13] F. Robert, G. Petel, Nucleotide synthesis capability as a marker of the petiole elongation of strawberry plants (Fragaria × ananassa Duch.), J. Hortic. Sci. Biotechnol. 75 (2001) 690–696. [14] J. Sacher, G. Towers, D.D. Davies, Effect of light and ageing on enzymes, particularly phenylalanine ammonia lyase, in discs of storage tissue, Phytochemistry 11 (1972) 2383–2391. [15] M. Shirzadegan, P. Christie, J.R. Seemann, An efficient method for isolation of RNA from tissue cultured plant cells, Nucleic Acids Res. 19 (1991) 6055. [16] C. Stasolla, R. Katahira, T.A. Thorpe, H. Ashihara, Purine and pyrimidine nucleotide metabolism in higher plants, J. Plant Physiol. 160 (2003) 1271–1295. [17] T. Takamura, I. Uritani, Changes in acid-soluble nucleotides in cutinjured sweet potato root tissue, Agric. Biol. Chem. 37 (1973) 1511– 1515. [18] M. Teramoto, C. Koshiishi, H. Ashihara, Wound-induced respiration and pyrophosphate: fructose-6-phosphate phosphotransferase in potato tubers, Z. Naturforsch. 55c (2000) 953–956.