Plant Physiol. (1999) 119: 455-462

In Vitro Biosynthesis of Phosphorylated Starch in Intact Potato Amyloplasts¹

Bente Wischmann^*, Tom Hamborg Nielsen, and Birger Lindberg Møller

Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark

	ABSTRACT

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Intact amyloplasts from potato (Solanum tuberosum L.) were used to study starch biosynthesis and phosphorylation. Assessed by the degree of intactness and by the level of cytosolic and vacuolar contamination, the best preparations were selected by searching for amyloplasts containing small starch grains. The isolated, small amyloplasts were 80% intact and were free from cytosolic and vacuolar contamination. Biosynthetic studies of the amyloplasts showed that [1-¹⁴C]glucose-6-phosphate (Glc-6-P) was an efficient precursor for starch synthesis in a manner highly dependent on amyloplast integrity. Starch biosynthesis from [1-¹⁴C]Glc-1-P in small, intact amyloplasts was 5-fold lower and largely independent of amyloplast intactness. When [³³P]Glc-6-P was administered to the amyloplasts, radiophosphorylated starch was produced. Isoamylase treatment of the starch followed by high-performance anion-exchange chromatography with pulsed amperometric detection revealed the separated phosphorylated -glucans. Acid hydrolysis of the phosphorylated -glucans and high-performance anion-exchange chromatography analyses showed that the incorporated phosphate was preferentially positioned at C-6 of the Glc moiety. The incorporation of radiolabel from Glc-1-P into starch in preparations of amyloplasts containing large grains was independent of intactness and most likely catalyzed by starch phosphorylase bound to naked starch grains.

	INTRODUCTION

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Starches from tuberous plants such as potato (Solanum tuberosum L.) contain a small fraction of covalently bound phosphate in their amylopectin (Jane et al., 1996). Approximately 60% to 70% of the phosphate groups are linked to C-6 of the Glc residues, 30% to 40% to C-3, and a small fraction (1%) may be linked to C-2 (Hizukuri et al., 1970). Bay-Smidt et al. (1994) detected variations in the level of starch phosphorylation among different potato varieties, and Abel et al. (1996) and Lorbert and Kossmann (1997) observed similar variations among transgenic potato lines with altered levels of the enzymes involved in starch biosynthesis. However, the mechanism responsible for potato starch phosphorylation remains elusive. Nielsen et al. (1994) used a potato tuber disc system to show that starch phosphorylation proceeds concomitantly with the de novo biosynthesis of starch. Starch is synthesized and stored in amyloplasts; the availability of isolated intact amyloplasts thus constitutes an important tool for studies of the phosphorylation process.

Intact amyloplasts have been purified from storage organs of a number of important crops and used to study the regulation of the carbon flux into starch (Echeverria et al., 1988; Entwistle et al., 1988; Mohabir and John, 1988; Smith et al., 1990; Tetlow et al., 1993; Kosegarten and Mengel, 1994; Naeem et al., 1997). Biosynthetic studies using different crops (pea, cauliflower, maize, and wheat) showed that amyloplasts import carbon from the cytosol in the form of hexose-P (Tyson and ap Rees, 1988; Hill and Smith, 1991; Neuhaus et al., 1993). This conclusion was strengthened by determining the degree of labeling randomization in starch that was isolated after incubation with Glc isotopically labeled at the C-1 or C-6 position (Keeling et al., 1988; Hatzfeld and Stitt, 1990; Viola et al., 1991). Entwistle and ap Rees (1990) showed that potato tubers lack plastidic Fru-1,6-bisphosphatase activity; Kossmann et al. (1992) cloned the gene encoding the plastidic Fru-1,6-bisphosphatase from leaves and determined that it was not expressed in potato tubers. Therefore, hexose-P, not triose-P, must be imported and used directly for starch synthesis in potato tuber amyloplasts. Two reports presented evidence that hexose-P is imported into the amyloplasts of potato tubers in the form of Glc-1-P (Kosegarten and Mengel, 1994; Naeem et al., 1997). A third report demonstrated that Glc-6-P is the preferred transported metabolite in proteoliposomes derived from potato tuber amyloplasts (Schott et al., 1995).

We have examined the ability of different potato tuber amyloplast preparations to import hexose-P and to synthesize phosphorylated starch. We now document that intact amyloplasts containing small starch grains efficiently import Glc-6-P and are able to perform de novo synthesis of phosphorylated starch.

	MATERIALS AND METHODS

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Plant Material and Chemicals

Preparation of Homogenate and Isolation of Intact Amyloplasts

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Amyloplasts were isolated from potato tuber pieces (approximately 2 g), which were individually chopped using a razor blade while positioned on a stainless steel mesh (300 µm) mounted in a small glass beaker, and submersed in the isolation medium (45 mL). The material remaining on the mesh after chopping was carefully removed before a new piece of tissue was processed in the same medium. Finally, the mesh was removed and the small amyloplasts were collected as the upper 12 mL of the suspension in the beaker. After removal of larger starch grains by a gentle centrifugation step (30g, 5 min, 4°C), an aliquot (500 µL) of the supernatant (Table I, crude extract) was used for enzyme assays. Two aliquots (5 mL) of the crude extract were applied to 5 mL of the isolation buffer with 2% (w/v) Nycodenz (Sigma) layered on a 1% (w/v) Bacto agar pad (Difco Laboratories, Detroit, MI) and subjected to centrifugation (30g, 25 min, 4°C). The amyloplasts on the agarose pad were gently resuspended in 5 mL of isolation medium, layered on 5 mL of 2% (w/v) Nycodenz, and recentrifuged as before. The recovered amyloplasts (Table I, small amyloplasts) were resuspended in a 2.1-mL (low amyloplast concentration) or a 0.25-mL (high amyloplast concentration) buffer and held for additional experiments. Grain size distribution of the small amyloplasts was determined by transmission electron microscopy. Large amyloplasts were obtained using the same procedure except that the bottom part of the suspension was used, and the initial centrifugation step was omitted.

Enzyme Assays

We monitored the marker enzyme activities according to the following previously reported procedures. Amyloplasts: AGPase (EC 2.7.7.27) was determined as described by Sowokinos (1976), except for the addition of 500 mM sorbitol to the assay mixture, alkaline inorganic pyrophosphatase (EC 3.6.1.1) was quantified spectrometrically (600 nm) as described by Gross and ap Rees (1986); cytosol: ADH (EC 1.1.1.1) and pyrophosphate:Fru-6-P phosphotransferase (EC 2.7.1.90) were determined as described by MacDonald and ap Rees (1983) and Nielsen et al. (1991), respectively; vacuole: -mannosidase (EC 3.2.1.24) was quantified spectrometrically (405 nm) as described by Stitt et al. (1989); mitochondria: Cyt c oxidase (EC 1.9.3.1) was quantified spectrometrically (550 nm) in the presence of 0.025% Triton X-100 (Rasmussen and Møller, 1990); and other enzymes: ATPase activity was quantified as the liberation of radiolabeled orthophosphate from [-³³P]ATP. The reaction mixture (total volume of 24 µL) contained amyloplasts (8 µL of a high concentration of amyloplasts), 66.6 kBq [-³³P]ATP, 1 mM ATP, 5 mM MgCl₂, 1 mM EDTA, and 50 mM Mops/KOH, pH 7.3. The reaction was terminated by applying aliquots (1 µL) of the reaction mixture onto a TLC plate (silica gel 60, Merck, Darmstadt, Germany). The products formed were separated by developing the TLC plate for 45 min in a solvent containing 35 mL of methanol, 15 mL of water, and 0.5 g of NaCl. The distribution of radiolabel was monitored by autoradiography. The regions corresponding to the positions of ATP and Pi were excised, resuspended in 1 mL of water mixed with 10 mL of Ecoscint A (National Diagnostics, Manville, NJ), and the radioactivity was quantified by liquid-scintillation counting (model 1215 RackBeta, Pharmacia LKB). The reaction was linear for at least 20 min.

Amyloplast Intactness

Assessment of Conversion between Glc-6-P and Glc-1-P

Starch Synthesis Using [¹⁴C]Glc-6-P or [¹⁴C]Glc-1-P as a Precursor

Starch Synthesis Using [³³P]Glc-6-P as a Precursor

Isolation of Phosphorylated Linear Oligosaccharides

The second half of the amyloplast starch sample was hydrolyzed with -amylase (Termamyl, Novo, Copenhagen, Denmark) as described by Nielsen et al. (1994). After a short centrifugation, the phosphorylated glucans produced were isolated by passing the supernatant over a DEAE-Sepharose column and freeze-dried. Some phosphoglucan fractions were further hydrolyzed in 0.7 N HCl (2-4 h, 100°C) to cleave all of the -glucosidic bonds present. They were freeze-dried, resuspended, and neutralized in a small volume before the generated content of Glc and Glc phosphates was determined by HPAEC-PAD.

HPAEC-PAD Analysis

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	RESULTS

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Amyloplast Characterization

The purity of the isolated amyloplasts with respect to the presence of other organelles was assessed by measuring the activity of marker enzymes for cytosol, mitochondria, and vacuoles. The data obtained in a typical set of experiments are shown in Table I. In this set, the total AGPase activity in the isolated, small amyloplasts was 11.7 nmol tuber¹ min¹. However, among individual preparations, the total AGPase activity varied from 3 to 30 nmol tuber¹ min¹, resulting in a mean value of 10 ± 7 nmol tuber¹ min¹ (mean ± SD, 23 preparations). A comparison of the amyloplast AGPase activity with that of other marker enzyme activities demonstrated that the isolated amyloplasts were not contaminated by other organelles except mitochondria. Compared with the preparations of small amyloplasts, the large amyloplast preparations had a much lower level of intactness (65%, mean of 14 experiments).

Starch Synthesis

View larger version (36K): [in this window] [in a new window]   Figure 1. Starch biosynthesis from [1-14C]Glc-6-P and [1-14C]Glc-1-P in experiments with ruptured () and intact () potato tuber amyloplasts. Small amyloplasts were selected for the experiment. The amyloplasts were ruptured by treatment with 0.1% Triton X-100. The amyloplasts were incubated for 1 h with 4 mM exogenously added ATP and 3PGA and 2 mM metabolite (Glc-6-P or Glc-1-P) with 6.3 kBq [1-14C]Glc-6-P or [1-14C]Glc-1-P. The amount incorporated was normalized on the basis of the activity of AGPase in the plastid preparations. Each column represents a mean of three replicates. Each separate amyloplast preparation is numbered using roman numerals. U, Units.

In contrast to the results obtained with the small amyloplasts, Glc-1-P was the preferred substrate for starch synthesis in the large amyloplasts. As observed for the small amyloplasts, the formation of starch from Glc-1-P was largely independent of amyloplast intactness (Fig. 2). The large amyloplasts showed a significantly higher rate of starch synthesis from Glc-1-P, whereas the synthesis from Glc-6-P was at a similar range as in the small amyloplasts.

View larger version (49K): [in this window] [in a new window]   Figure 2. Starch biosynthesis from [1-14C]Glc-6-P and [1-14C]Glc-1-P in experiments with ruptured () and intact () plastids prepared from large amyloplasts. The amyloplasts were incubated as described in the legend to Figure 1. Each set of experiments is numbered. The amount of hexose residues incorporated is calculated as described in the legend to Figure 1. U, Units.

Small Amyloplasts de Novo Synthesize Phosphorylated Starch

View larger version (39K): [in this window] [in a new window]   Figure 3. Verification of incorporated phosphate into starch in experiments with small amyloplasts using [33P]Glc-6-P and effect of -amylase. A, HPAEC elution profile of linear phosphorylated oligosaccharides together with the corresponding radioactivity. B, Detector response and corresponding radioactivity after -amylase treatment of the linear phosphorylated oligosaccharides. In both panels peaks of radioactivity were integrated and normalized to 100%. nC, Nanocoulombs.

The elution profile obtained after -amylase treatment of the ³³P-labeled phosphorylated -glucans and HPAEC-PA1 column chromatography is shown in Figure 4A. Using the PA1 column, Glc-6-P eluted at 25 mL (Fig. 5C) and Glc-3-P at 27 mL (Fig. 5B), whereas the short phosphorylated -glucans eluted between 25 and 33 mL (Fig. 5A). When the preparation of phosphorylated short -glucans was acid hydrolyzed to cleave all -glucosidic linkages, we expected the incorporated ³³P to be recovered in Glc-6-P and Glc-3-P, with Glc-6-P as the predominant component (Bay-Smidt et al., 1994). The radioactivity profile obtained was in agreement with this interpretation (Fig. 4B). After -amylase degradation, acid hydrolysis, and PA1 chromatography, 79% of the radioactivity was recovered as hexose-P. Radiolabeled Glc-6-P was the major component; low amounts of radioactivity co-eluted with Glc-3-P.

View larger version (29K): [in this window] [in a new window]   Figure 4. Verification of phosphate incorporated into starch at C-6 and C-3 in the Glc residues in experiments with small amyloplasts using [33P]Glc-6-P. A, HPAEC elution profile of phosphorylated dextrins treated with -amylase and corresponding radioactivity. B, Detector response and radioactivity after acid hydrolysis: 2 h in 0.7 N HCl at 100°C. nC, Nanocoulombs.

View larger version (30K): [in this window] [in a new window]   Figure 5. HPAEC elution profile of phosphorylated acid-hydrolyzed dextrins, as in Figure 4B, showing the elution time of internal standards of Glc-3-P and Glc-6-P. A, No internal standard added; B and C, Glc-3-P and Glc-6-P added as internal standards, respectively. nC, Nanocoulombs.

	DISCUSSION

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Potato tuber amyloplasts with an intactness of 80% were isolated using mechanical disruption of the tuber tissue in medium containing 0.5 M sorbitol followed by density gradient purification. The amyloplasts remained intact for several hours at room temperature. Amyloplast preparations were essentially free of cytosolic and vacuolar enzyme activities (Table I).

The isolation of potato amyloplasts has previously been reported based on tissue culture material derived from tubers (Mohabir and John, 1988; Kosegarten and Mengel, 1994) or on the use of tuber tissue as the starting material (Mohabir and John, 1988; Schott et al., 1995; Naeem et al., 1997), as in this study. The yield determined from the relative activity of the amyloplast marker enzyme AGPase (Table I) was low (0.4% of the amyloplasts released from the tissue) compared with the 10% yield reported by Naeem et al. (1997). However, all attempts to increase the yield compromised the ability to obtain amyloplasts with high intactness and purity and reflected the selection for small amyloplasts.

Because triose-P cannot be converted to hexose-P inside potato tuber amyloplasts and therefore cannot be used directly for starch synthesis (Entwisle and ap Rees, 1990; Kossmann et al., 1992), we studied starch synthesis using Glc-6-P and Glc-1-P as the substrates and demonstrated that intact, small potato amyloplasts could import and use Glc-6-P for the synthesis of starch (Fig. 1). In proteoliposomes reconstituted from potato amyloplasts isolated from transgenic potato with a reduced starch content, Glc-6-P, but not Glc-1-P, was translocated by counterexchange with Pi, dihydroxyacetonephosphate, and 3PGA (Schott et al., 1995). Others have reported that potato amyloplasts preferentially took up Glc-1-P for starch synthesis (Naeem et al., 1997). Kosegarten and Mengel (1994) also showed starch synthesis from Glc-1-P, but did not test Glc-6-P as a substrate. The differences concerning the preferred precursor for import and subsequent synthesis of starch may be explained by the different methods used for plastid isolation.

The data in this study clearly demonstrated that Glc-6-P was the precursor for starch synthesis in small amyloplasts and that only intact amyloplasts contributed to the incorporation. In contrast to these results, Glc-1-P was the most efficient precursor for starch synthesis when large amyloplasts were used (Fig. 2). The incorporation from Glc-1-P proceeded independently of amyloplast intactness. The preparation of large amyloplasts contained a large fraction of "naked" starch grains. Several of the enzymes involved in starch biosynthesis, including starch phosphorylase, are known to be associated with starch grains (Martin and Smith, 1995). The high rate of starch biosynthesis observed upon administration of Glc-1-P to large amyloplasts, as in the present study, may therefore be explained by the activity of starch phosphorylase bound to contaminating naked starch grains. This is not a plausible explanation, however, for the conflicting results obtained by Naeem et al. (1997), who found that starch biosynthesis from Glc-1-P was increased by the addition of ATP and was dependent on intactness. An alternative explanation is that small potato amyloplasts use Glc-6-P and large potato amyloplasts use Glc-1-P as a precursor for starch synthesis, although no physiological arguments are available to support such a difference.

Maize endosperm amyloplasts incorporated both Glc-1-P and Glc-6-P at comparable rates when supplied at high concentrations (above 2 mM) (Neuhaus et al., 1993). However, only Glc-6-P was imported via a counterexchange in proteoliposomes from maize endosperm (Möhlmann et al., 1997). Schott et al. (1995) obtained similar results with proteoliposomes derived from a potato plastid envelope where counterexchange with Pi was tested. The rate of starch synthesis from Glc-6-P obtained with the small amyloplasts used in this study varied from experiment to experiment. The rates (Fig. 1) are comparable to the rate of starch synthesis (4.9 µmol hexoses incorporated h¹ unit¹ AGPase) reported by Naeem et al. (1997) using Glc-1-P as a precursor.

A remarkable feature of potato starch is its content of phosphate esterified to C-6 and C-3 in the Glc moieties of the amylopectin molecules. Here we show that starch synthesized in vitro by isolated potato amyloplasts is phosphorylated.

The elution of the phosphorylated glucans obtained by the debranching of amylopectin depends on the length of the glucan chain, the number of phosphate groups esterified to each glucan chain, the position of the phosphate in the chain, and probably also on the position of the phosphate on the Glc residues (Blennow et al., 1997). -Amylolysis of phosphorylated glucans releases maltose from the nonreducing end of the glucan (Takeda and Hizukuri, 1981). The -amylase cannot bypass Glc residues esterified with phosphate at the C-6 or C-3 position, and the -glucans recovered after -amylase treatment are therefore expected to be shortened to chains representing the distance from the former amylopectin branchpoint to the phosphate group on the glucan chain. In agreement with this assumption, -amylolysis produced a shift toward shorter glucans. Similarly, -amylase-treated and debranched phosphorylated glucans eluted within the first 20 mL (B. Wischmann, unpublished results).

Analysis using HPAEC-PAD programs that were optimized to separate Glc-6-P from Glc-3-P showed that the radioactivity obtained after acid hydrolysis was concentrated at the position of Glc-6-P and to a lesser degree at the position of Glc-3-P (compare Figs. 4B and 5). We therefore conclude that the de novo-synthesized starch is phosphorylated at the C-6 position but only secondarily at the C-3 position.

In conclusion, we have isolated intact amyloplasts containing small starch grains. These intact amyloplasts use Glc-6-P as an efficient precursor for starch synthesis, and the de novo-synthesized starch is phosphorylated at the C-6 position and to some extent also at the C-3 position of the Glc residues.

	FOOTNOTES

   Received August 3, 1998; accepted October 13, 1998.

	ABBREVIATIONS

Abbreviations: ADH, alcohol dehydrogenase. AGPase, adenosine-5 diphosphoglucose pyrophosphorylase. HPAEC-PAD, high-performance anionic-exchange chromatography with pulsed amperometric detection. 3-PGA, 3-phosphoglyceric acid.

	ACKNOWLEDGMENTS

We gratefully acknowledge Dr. Alison M. Smith (John Innes Centre, Norwich, UK) for her help and advice during the initial isolations of intact potato amyloplasts. We also thank Bjarne Mejer for excellent technical assistance. Glc-3-P was a kind gift from Dr. Francis Kappler (Fox Chase Cancer Center, Philadelphia, PA).

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