Plant and Cell Physiology Advance Access originally published online on September 4, 2008
Plant and Cell Physiology 2008 49(10):1572-1579; doi:10.1093/pcp/pcn127
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A Cell Wall-Bound Adenosine Nucleosidase is Involved in the Salvage of Extracellular ATP in Solanum tuberosum
1 Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany
2 Leibniz-Institute of Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1, D-14979 Grossbeeren, Germany
*Corresponding author: E-mail, geigenberger{at}igzev.de; Fax, +49-33701-55391.
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Abstract |
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Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.
Keywords: Adenosine nucleosidase - Apoplast - Apyrase - Cell wall - Extracellular ATP - Solanum tuberosum
Abbreviations: ANase, adenosine nucleosidase; CCCP, carbonyl cyanide m-chloro phenyl hydrazone; eATP, extracellular ATP; GC-MS, gas chromatography–mass spectrometry; UGPase, UDP-glucose pyrophosphorylase; PNP, p-nitrophenylphosphate; PUP, purine permease; RNAi, RNA interference.
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Introduction |
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In plants, growing evidence has emerged that extracellular ATP (eATP) acts as a signaling molecule on the surface of plant cells. Manipulation of eATP by application of either ATP or ATP-consuming enzymes led to responses that suggest eATP having a function in stress and wound responses (Jeter et al. 2004
, Song et al. 2006), gravitropic growth (Tang et al. 2003) and regulation of cell viability (Chivasa et al. 2005). Visualization of eATP in planta with a recombinant cellulose-binding luciferase revealed that eATP is present predominantly in growing and expanding regions (Kim et al. 2006).
Plants also contain ATP diphosphohydrolases (apyrases) in the extracellular compartment which convert ATP to ADP and AMP. Apoplastic localization of apyrase has been clearly documented for Medicago truncatula (Day et al. 2000) and more recently for Solanum tuberosum (Riewe et al. 2008a). Transgenic reduction of apyrases in Arabidopsis or potato led to reduced growth and altered development, providing genetic evidence that apoplastic apyrase activity is necessary for growth (Wu et al. 2007, Riewe et al. 2008a). This has been confirmed by expression studies, showing that apyrases are preferentially expressed in growing regions of potato (Riewe et al. 2008a) and Arabidopsis plants (Wu et al. 2007), On the basis of these results, a model has been proposed that links apyrase to growth via regulation of eATP. It has been suggested that eATP is transmitting its signal either via an as yet unidentified plasma membrane receptor (Jeter et al. 2004, Roux and Steinebrunner 2007), by binding to a soluble receptor or by protein phosphorylation (Chivasa et al. 2005).
Despite the emerging role of eATP in plant signaling, little is known about how ATP is transmitted to the apoplast, and how eAMP is further metabolized to be re-imported into the cell. As possible sources of eATP, osmotic stress, exocytosis, loss of plasma membrane integrity or wounding have been suggested (Roux and Steinebrunner 2007). Moreover, Thomas et al. (2000) showed that eATP increased in leaves overexpressing the ABC transporter MDR1, providing evidence that eATP is released by ABC transporters. Once eATP has been converted to eAMP by apyrase in the apoplastic space, eAMP, or a breakdown product thereof, has to be salvaged by re-import into the cell. Since no plasmalemma AMP transporters have been characterized yet, it is assumed that AMP is hydrolyzed to adenosine and Pi by a phosphatase. In potato tubers, a phosphatase bound to the cell wall has been identified by enzymatic release from cell walls of intact tuber slices (Stephens and Wood 1974) and activity measurements of insoluble tuber cell material (Tu et al. 1988). Unfortunately, only p-nitrophenyl phosphate (PNP) was tested in both studies, so that no information on the specificity of this enzyme towards nucleotides, in particular AMP, is available for potato.
Adenosine could be transported into the cell by equilibrative nucleoside transporters (Mohlmann et al. 2001), a class of transporters that so far have been characterized to function partially as H+ symporters or independently of H+. It has been hypothesized by Wormit et al. (2004) that these transporters may be involved in a pathway salvaging eATP. Plants also posses purine permeases (PUPs), which transport adenine into the cell along a H+ gradient (Gillissen et al. 2000, Burkle et al. 2003). Theoretically, apoplastic adenosine could be further converted to adenine by an apoplastic nucleosidase and reimported by PUPs. This model is supported by the finding that insoluble material from cabbage leaf, which consists mainly of cell wall, contains adenosine-specific nuclease activity (Mazelis 1959), even though this activity was not considered to be in the cell wall by the author.
To investigate the fate of eATP in the apoplast, we first incubated intact potato tuber slices with ATP, ADP, AMP and adenosine, and analyzed the reaction products using HPLC, gas chromatography–mass spectrometry (GC-MS), colorimetric and spectrophotometric assays. We then determined import rates of radiolabeled adenine in the presence of these adenylates. Incubation with ATP led to the formation of ADP, AMP, adenosine, adenine and Pi, and an inhibition of adenine uptake. In addition to the quantification of the already described apoplastic activities such as apyrase and unspecific phosphatase carrying out 5'-nucleotidase activity, this led to the discovery of a cell wall-bound nucleosidase activity. This nucleosidase was highly adenosine specific and thus termed adenosine nucleosidase (ANase). By correlation analysis, we found significant evidence that ANase activity is tightly co-regulated with apyrase activity in wild-type tubers. Analysis of tubers from transgenic plants with strongly reduced apyrase activity showed that these plants had reduced ANase activity too. On the basis that ANase converts a downstream product of apyrase, the apoplastic localization of both enzymes and their coordinated activity, we suggest that apyrase and ANase are functionally linked in an apoplastic adenylate salvage pathway.
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The potato tuber apoplast converts ATP into adenine, ribose and Pi
To obtain insight into the apoplastic metabolism of ATP, we incubated intact tuber slices from 8-week-old potato plants in buffer containing 1 mM ATP. Samples were taken over a time course and analyzed by HPLC. The elution chromatogram (Fig. 1A) clearly revealed that ATP was converted into ADP, AMP, adenosine and adenine within the time points chosen for analysis. By incubating the slices with 1 mM AMP instead of ATP (Fig. 1B), we could demonstrate that both adenosine and adenine were produced from AMP. Incubation with 1 mM adenosine suggested that adenine is produced from adenosine (Fig. 1C). To confirm the identity of the adenosine and adenine peaks, we spiked some samples with adenosine and adenine (Fig. 1C, D). If adenosine was converted into adenine, ribose should be produced in parallel. For this reason, aliquots from tuber slice incubations were also analyzed using GC-MS. The only sugar found in considerable amounts in these incubation extracts was ribose (Fig. 1E). The ratio of ribose to adenine was approximately 1 : 1, indicating operation of a nucleosidase (see Supplementary Fig. S1). Due to instability of adenine after the derivatization step used in the GC-MS protocol, we chose ribose for quantitative analyses. The combined results of these studies clearly suggest that the potato tuber apoplast possesses the necessary machinery to catalyze the conversion of ATP into adenine, ribose and Pi.
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In order to analyze whether ATP can be re-imported as adenine, we studied the inhibition of adenine uptake in the presence of ATP, ADP or AMP. Zeatin was chosen as positive control, since it has been reported that PUP transporters also transport this adenine derivative (Burkle et al. 2003
). As a negative control, sucrose was applied instead of an adenylate/nucleobase. As is clearly visible in Fig. 1F, the presence of ATP, ADP, AMP and zeatin (all 500 µM) clearly led to a marked competitive inhibition of adenine uptake, whereas sucrose did not lead to an inhibition (data not shown). Further experimentation revealed that this transport was likely to be H+ dependent, since the presence of the ionophore carbonyl cyanide m-chloro phenyl hydrazone (CCCP) led to a strong inhibition of uptake (data not shown).
In addition to apyrase and phosphatase, the potato tuber apoplast contains a cell wall-bound nucleosidase
To confirm the presence of the enzymatic machinery capable of converting ATP to adenine, ribose and Pi within the plant apoplast, we compared the respective activities in intact tuber slices, total enzyme extracts and washed pellets of enzyme extracts containing enzymes associated with insoluble material such as cell wall or starch granules. As all activities were assayed in extracts from identical tuber material, this provides information on the relative contribution of the activities in the apoplast, the cell wall and the soluble fraction to the total cellular activity. In order to determine apoplastic nucleotide-hydrolyzing activities, five potato tuber slices were incubated with buffer containing ATP, AMP or the common phosphatase substrate PNP. After several time intervals, aliquots were collected and the levels of Pi, nucleotide and nitrophenol were analyzed. To assay adenosine-hydrolyzing activity, five slices were incubated with adenosine alone and in the presence of the ionophore CCCP to inhibit substrate/product uptake into the intact slices. Aliquots were collected at intervals during the incubation, and the production of ribose was monitored. For all measurements, linearity of product production over time was observed, and for UDP-glucose pyrophosphorylase (UGPase), apyrase and 5'-nucleotidase additional measurements without substrate were carried out to confirm that product formation was dependent on enzyme activity (Fig. 1E; Supplementary Fig. S2). All activities were also determined in both total enzyme extracts and washed insoluble pellets of these enzyme extracts (Table 1). The activity of UGPase in the intact tuber slices was <0.3% of the total activity, confirming that these preparations were essentially free of cytosolic activities. In accordance with previous experiments (Riewe et al. 2008a), apyrase activity detected in tuber slices was approximately 6% of the total activity, with the activity being much higher in the extract than in the pellet fraction.
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5'-Nucleotidase activity in the intact tuber slices represented 10% of overall activity, suggesting that the apoplast contains a significant activity of this type. 5'-Nucleotidase in the intact tuber slices did not appear to be specific for nucleoside monophosphates, since PNP was hydrolyzed with only slightly higher speed (18.6 ± 0.5 mU g FW–1, data not shown) than AMP (13 ± 0.3 mU g FW–1, see also Table 1). Whereas in intact tuber slices ATP was hydrolyzed much faster than AMP due to activity of apyrase (see Table 1), in the pellet fraction hydrolysis of ATP (55 ± 12 mU g FW–1) and AMP was equal (58 ± 7 mU g FW–1, see also Table 1) and PNP hydrolysis was only slightly higher (83.4 ± 8.4 U g FW–1, data not shown), pointing to a low substrate specificity of the enzyme. From this we conclude that the eAMP was hydrolyzed by an unspecific cell wall-bound phosphatase rather than by a specific 5'-nucleotidase
As expected from the preceding experiments, a high nucleosidase activity was found in the intact tuber slices. The activity presented in Table 1 (10 ± 1.6 mU g FW–1) was obtained in experiments using 50 µM CCCP to inhibit H+-dependent ribose uptake, but was only slightly lower without the ionophore (8.9 ± 0.4 mU g FW–1). This activity contributed to 15% of the total activity. The pellet contained 99% of total nucleosidase activity, suggesting that this enzyme is bound to the cell wall, hardly present in the cytosol and not released from the cell wall under the conditions of extraction used here. The low abundance of nucleosidase activity in the soluble fraction of the enzyme extract is in close agreement with recently published work on potato tubers measured via a radiochemical assay (Katahira and Ashihara 2006). Adenine uptake was determined to be 0.6 mU g FW–1 (one order of magnitude lower than nucleosidase activity). This observation is in keeping with the observed accumulation of adenine if adenosine (or indeed any adenylate) is offered to tuber slices (see Fig. 1A–C). When taken together, these experimental data provide evidence for the existence of a series of three enzymes that are capable of converting ATP to adenine, ribose and Pi. In a similar experiment, Mazelis (1959) described enzymatic activities converting ATP to adenine in insoluble particles of enzyme extracts prepared from cabbage leafs almost 50 years ago. Mazelis, however, argued for a cytosolic localization of both the insoluble particles and their attached activities.
The cell wall-bound nucleosidase is highly specific for adenosine
In our previous experiments, nucleosidase activity was assayed using adenosine as substrate. To analyze the biochemical properties of this activity further, several putative substrates with structural similarity to adenosine were analyzed. For this purpose, we tested the purine nucleosides guanosine and inosine, as well as the pyrimidine nucleosides cytidine, uridine and thymidine, and the phytohormone zeatin riboside. There is considerable indirect evidence for turnover of cytokinin ribosides in plants; however, the enzyme(s) responsible have, as yet, not been identified (Auer 2002). Using the same experimental set-up as defined above, washed intact tuber slices and washed pellets were incubated with different putative substrates, and ribose production was monitored by a Fehling-based assay. In both tuber slices and pellets, ribose was produced following incubation with adenosine but not with uridine, thymidine, cytidine, guanosine, inosine or zeatin riboside (Fig. 2A, B). These results clearly demonstrate that the cell wall-bound nucleosidase activity is highly specific for adenosine and can therefore be termed adenosine nucleosidase (ANase).
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The cell wall-bound adenosine nucleosidase is functionally linked to apyrase
Recently, we have shown that the potato-specific apyrase is confined to the apoplast (Riewe et al. 2008a
). Given that we have now identified a cell-wall bound ANase, it is conceivable that nucleosidase might act together with apyrase and a non-specific phosphatase in a pathway to convert eATP into adenine. Given that constituent enzymes of a pathway are often co-regulated at the transcript, protein or activity level, as has been shown for glycolytic or tricarboxylic acid cycle enzyme activities (Mitchell-Olds and Pedersen 1998, Cross et al. 2006), we chose to assess whether this was the case for these enzymes. For this purpose, we measured apyrase and ANase in individual tubers of different developmental stages from different plants in two independent experiments (n = 18 and 6, respectively). As shown in Fig. 3A, the correlation between ANase and apyrase was highly significant (r = 0.81, P = 2 x 10–6, n = 24). However, neither ANase nor apyrase correlated with the cytosolic enzyme UGPase (Fig. 3B, r = 0.06, P = 0.82, n = 18; and data not shown) or the protein content of the enzyme extract (r = 0.11, P = 0.67, n = 18 for ANase vs. protein content; and data not shown). The strong and specific correlation between ANase and apyrase strongly favors the hypothesis that these enzymes are constituents of the same pathway.
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To characterize the coordination of these activities further, we assayed ANase and apyrase activities in tuber tissue from transgenic potato plants with reduced (B33:25 and 35S:23) or elevated (35S:113) apyrase activity (Fig. 3C). As already described previously (Riewe et al. 2008a
), apyrase activity in intact slices was significantly reduced in the RNA interference (RNAi) lines B33:25 and 35S:23 to a level of 43–45% of the wild-type, and increased in the overexpressor line 35S:113 to approximately 500%. Irrespective of whether assayed in intact tuber slices or pellets, ANase was significantly reduced to 28–60% of the wild-type activity in both RNAi lines. This was, however, not the case for the overexpressor line 35S:113. In this line, ANase was not significantly altered when compared with the wild type. These data demonstrate that strong reduction of apyrase leads to a reduction of ANase. We can exclude that ANase activity is performed by apyrase, because ANase is not altered in the apyrase-overexpressing line. |
Discussion |
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Our results provide evidence for an apoplastic salvage pathway for eATP in potato tubers, consisting of apyrase, unspecific phosphatase, ANase and an adenine transport system (see model in Fig. 4). Using intact tuber slices as a model system for the potato tuber apoplast, we were able to assay apyrase, phosphatase and an as yet uncharacterized ANase. Since all activities were measured from an identical source in a single experiment, comparison between the absolute activities of these enzymes in the apoplastic space of potato tubers was possible. 5'-Nucleotidase and ANase activities were almost identical, whilst apyrase activity (when assayed on the basis of the conversion of ATP to AMP instead of Pi release) was higher but of a similar magnitude (Table 1).
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Apyrase has been shown to be a soluble apoplastic protein enclosed by the cell wall (Riewe et al. 2008a
), but we cannot currently exclude the possibility that it is weakly associated with the cell wall or cell membrane (see model, Fig. 4). Free diffusion or related kinetic effects after extraction might explain its relatively high activity in the soluble fraction when compared with the activity determined in intact tuber slices. Unspecific phosphatase and ANase were found to be more stringently bound to the cell wall and were not released under native extraction procedures including the non-ionic detergent Triton X-100. The cell wall-bound phosphatase revealed a broad substrate spectrum in accordance with those cell wall phosphatases characterized from other plant species such as mustard (Duff et al. 1991) or white clover (Zhang and McManus 2000). The nucleosidase identified in the apoplast was highly specific for adenosine, and this implies that adenosine is converted into adenine in vivo and subsequently imported into the cell by an adenine transporter. Our data show that ATP (or ADP and AMP) leads to a competitive inhibition of adenine uptake into potato tuber slices. It is unlikely that this competition is direct, because adenine transport by the Arabidopsis purine permease 1 was not inhibited by adenylates at all and only to a low extent by adenosine (Gillissen et al. 2000). Although speculative, it seems more likely that inhibition was caused by adenine, which was produced by concerted action of apyrase, phosphatase and ANase. Under saturating conditions, AMP or adenosine are converted in significant amounts into adenine by intact tuber slices without relevant diminution of the sum of AMP, adenine and adenosine by uptake processes within 240 min (see peak areas in Fig. 1B and C). If this applies to the situation in vivo, ANase would bypass adenosine uptake, and adenine uptake would take place instead (see Fig. 4).
A further indication for an involvement of ANase in eATP salvage is given by the fact that apyrase and ANase are coordinately regulated at the activity level (Fig. 3). We found a high and significant co-regulation of apyrase and ANase activity in wild-type tubers of different developmental stages. This may be achieved either by shared transcriptional control or by sensing of one of the substrates/products of apyrase, phosphatase or ANase triggering transcriptional or post-transcriptional regulation. The latter scenario may partially explain why reduction of ANase is observed in transgenic lines with decreased apyrase activity (Fig. 3).
In conclusion, our results show the presence of an ANase in the apoplast of potato tuber parenchyma cells that is likely to be involved in an eATP salvage pathway.
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HPLC
A deep-frozen aliquot of 80 µl was mixed and thawed with 80 µl of 10% (w/v) trichloroacetic acid to stop any enzymatic activity, neutralized with 5 M KOH and diluted to a final volume of 1,600 µl with distilled water. After centrifugation, 20 µl were analyzed as described previously (Geigenberger et al. 1997
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GC-MS
A deep-frozen aliquot of 80 µl was lyophylized, resolved in 1,600 µl of methanol and centrifuged. A 100 µl aliquot of the supernatant was evaporated, derivatized and analyzed as described by Roessner et al. (2001). The system was calibrated using ribose from 5 to 5,000 pmol per derivatized sample.
Adenine uptake rates
Freshly prepared tuber slices were washed and incubated with 20 mM MES/KOH, pH 6.0. Adenine was applied in concentrations ranging from 0.25 to 500 µM and 200 Bq of [U-14C]adenine (GE Healthcare, Uppsala, Sweden) alone or together with the potential inhibitor in 500 µM concentration in a final volume of 300 µl of buffer. After 75 min of gentle shaking, the slices were twice briefly washed in buffer. After homogenization, incorporated radioactivity was determined.
Preparation of intact slices, soluble enzyme extracts and pellets for enzyme analysis
Freshly harvested potatoes were sliced using a corkborer (diameter 8 mm) and a sharp knife The slices were either immediately frozen in liquid N2 or washed four times with 20 mM MES/KOH pH 6.0, 1 mM CaCl2. Five slices were transferred to 3 ml of assay buffer for measurement of the enzymatic activity specified below. The N2-frozen slices were homogenized using a MM301 Retsch mill (Retsch, Haan, Germany). Soluble proteins were extracted as previously described (Riewe et al. 2008b) and kept at 4°C prior to assay. The pellets of these extracts were washed one more time with extraction solution and twice with assay buffer. Then the pellet was subsequently suspended in a final volume of 2 ml of assay solution.
Enzyme analysis
UGPase. In the soluble extract, UGPase was assayed as described by Zrenner et al. (1993) in a 96-well Anthos ht3 photometer (ASYS Hitech, Eugendorf, Austria) in 200 µl pf assay mix containing 10 µl of 1 : 1,000 diluted enzyme extract. Intact tuber slices were incubated with assay mixture in a final volume of 3 ml on a shaker. After addition of PPi to a final concentration of 1 mM, aliquots of 200 µl were subtracted after short time intervals, and NADPH production was determined in a photometer as described for the soluble extracts. Analogously, the pellets were suspended in 2 ml of assay mixture and kept on a roller (Karl Hecht KG, Sondheim, Germany). After addition of PPi in a final concentration of 1 mM, 250 µl aliquots were subtracted in short intervals, briefly centrifuged and immediately analyzed in the photometer as described above.
Apyrase and 5'-nucleotidase. In soluble extracts, apyrase/5'-nucleotidase were measured by determination of Pi liberation as described previously (Riewe et al. 2008a) using 70 µl of 20 mM MES/KOH, pH 6.0, 1 mM CaCl2, 10 µl of extract and 2 µl of the substrates ATP or AMP in a final concentration of 1 mM. One unit was defined as 1 µmol Pi released per minute. Intact tuber slices were incubated with ATP or AMP in 1 mM concentration in a final volume of 3 ml of buffer on a shaker. After certain time points, 80 µl of buffer solution was subtracted and tested for Pi content or deep frozen and analyzed regarding nucleotide content using HPLC. The formation of hydrolyzed substrate was converted into Pi liberation. The pellet fraction was incubated with ATP or AMP in 1 mM concentration in a volume of 2 ml of buffer on a roller, and Pi formation was measured in briefly centrifuged 80 µl aliquots collected along a time course.
Unspecific phosphatase. In the soluble extract, unspecific phosphatase was assayed using the discontinuous assay with PNP as substrate (Bergmeyer 1985). A 10 µl aliquot of enzyme extract was incubated in 200 µl of 20 mM MES/KOH pH 6.0, 1 mM CaCl2. The reaction was started by addition of PNP to a final concentration of 1 mM. The reaction was terminated by the addition of 5 µl of 3 M NaOH to enable extinction of p-nitrophenol, and extinction at 405 nm was immediately determined in the photometer. If a precipitate occurred, the samples were centrifuged for 1 min at maximal speed, and 100 µl of the supernatant were read in the photometer. Intact tuber slices were incubated in 3 ml of the same buffer on a shaker and the reaction was started with PNP in a final concentration of 1 mM. Samples of 200 µl were subtracted from the buffer along a time course and analyzed as described for the soluble extracts. The pellets were suspended in the same buffer in a final volume of 2 ml and the samples were kept on a roller. The reaction was started with PNP in a final concentration of 1 mM. Samples of 250 µl were subtracted along a time course, centrifuged and analyzed as described above.
Nucleosidase. In soluble extracts nucleosidase was assayed using 1,900 µl of 20 mM MES/KOH, pH 6.0, 1 mM CaCl2, 100 µl of soluble extract, and started by addition of adenosine in a final concentration of 1 mM. Along a time course, 80 µl aliquots were subtracted and deep frozen, and ribose production was analyzed with GC-MS. Intact tuber slices were incubated in 3 ml of buffer on a shaker and the reaction was started by addition of nucleoside (adenosine, uridine, thymidine, cytidine, guanosine or zeatin riboside) in a final concentration of 1 mM. Along a time course, either 80 µl aliquots were subtracted, deep-frozen and analyzed with GC-MS (only adenosine) or 150 µl aliquots were subtracted and analyzed using a Fehling assay described by Parkin (1996). The pellets were suspended in 2 ml of assay buffer and incubated with nucleoside (adenosine, uridine, thymidine, cytidine, guanosine, inosine or zeatin riboside) in a final concentration of 1 mM on a roller. Aliquots were subtracted from the reaction mix and either analyzed using GC-MS or centrifuged and analyzed using the Fehling assay.
Supplementary material
Supplementary material are available at PCP Online.
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The Deutsche Forschungsgemeinschaft (grant GE 878).
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We wish to thank Gareth Catchpole and Aenne Eckard (MPI Molecular Plant Physiology, Potsdam, Germany) for their help with analyzing the GC-MS samples.
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(Received July 11, 2008; Accepted August 25, 2008)
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