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First published online March 19, 2004; 10.1104/pp.103.038026 Plant Physiology 134:1347-1354 (2004) © 2004 American Society of Plant Biologists Repression of a Novel Isoform of Disproportionating Enzyme (stDPE2) in Potato Leads to Inhibition of Starch Degradation in Leaves But Not Tubers Stored at Low Temperature1Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany (J.R.L.); Plant Research Department, Risø National Laboratory, DK–4000 Roskilde, Denmark (J.R.L., K.B., J.K.); and Plant Biochemistry Laboratory, Department of Plant Biology, Center for Molecular Plant Physiology, The Royal Veterinary and Agricultural University, DK–1871 Frederiksberg C, Copenhagen, Denmark (A.B.)
A potato (Solanum tuberosum) cDNA encoding an isoform of disproportionating enzyme (stDPE2) was identified in a functional screen in Escherichia coli. The stDPE2 protein was demonstrated to be present in chloroplasts and to accumulate at times of active starch degradation in potato leaves and tubers. Transgenic potato plants were made in which its presence was almost completely eliminated. It could be demonstrated that starch degradation was repressed in leaves of the transgenic plants but that cold-induced sweetening was not affected in tubers stored at 4°C. No evidence could be found for an effect of repression of stDPE2 on starch synthesis. The malto-oligosaccharide content of leaves from the transgenic plants was assessed. It was found that the amounts of malto-oligosaccharides increased in all plants during the dark period and that the transgenic lines accumulated up to 10-fold more than the control. Separation of these malto-oligosaccharides by high-performance anion-exchange chromatography with pulsed-amperometric detection showed that the only one that accumulated in the transgenic plants in comparison with the control was maltose. stDPE2 was purified to apparent homogeneity from potato tuber extracts and could be demonstrated to transfer glucose from maltose to oyster glycogen.
Disproportionating enzyme (D-enzyme; 4-  -glucanotransferase, EC 2.4.1.25) catalyzes the transfer (disproportionation) of -1,4 bonds between different glucans. It is located in plastids and has always been presumed to be involved in starch metabolism. Mutant and transgenic plants, which lack its activity almost entirely, have been isolated (Takaha et al., 1998 ; Colleoni et al., 1999a, 1999b; Critchley et al., 2001; Wattebled et al., 2003), and based on studies on these, two contradictory proposals have been made as to its role. Following analysis of the Chlamydomonas reinhardtii sta11 mutant, it was suggested that D-enzyme is involved directly in the synthesis of the amylopectin fraction of starch (Colleoni et al., 1999a, 1999b; Wattebled et al., 2003); however, no evidence for this could be found in an Arabidopsis D-enzyme mutant (dpe1; Critchley et al., 2001). It was demonstrated in this mutant that starch degradation was impaired, and it was proposed that the main role of D-enzyme is in malto-oligosaccharide metabolism during mobilization of starch in the dark period. In addition, transgenic potato (Solanum tuberosum) plants have been produced in which 99% of D-enzyme activity was repressed, but no influence on either amylopectin synthesis or starch degradation was reported (Takaha et al., 1998).
In all of the above studies, only one isoform of D-enzyme was repressed. Analysis of the Arabidopsis genome, however, has revealed that it contains two genes that appear to code for different D-enzyme isoforms. The mutation studied by Critchley et al. (2001)
Identification of cDNAs Coding for Proteins Capable of Metabolizing Maltose
In an effort to identify plant proteins capable of catabolizing maltose, the Escherichia coli TSM90 mutant was obtained from the CGSC: E. coli Genetic Stock Center. This strain cannot metabolize maltose, but it is not clear which gene is mutated that causes this phenotype. According to information provided by the CGSC stock center, the lesion lies in either the MalP (coding for maltodextrin phosphorylase), or the MalQ (coding for amylomaltase) gene. The TSM90 strain demonstrates a negative phenotype when grown on MacConkey agar supplemented with maltose, unless expressing a protein that gives it the ability to metabolize maltose (Ehrmann and Vogel, 1998
It should be stressed that this screen was not established specifically to identify D-enzyme isoforms, although that was the result. Indeed, in principle it should not have been possible to identify the stDPE1 isoform in this system, as it is thought not to be able to utilize maltose as a donor substrate (Jones and Whelan, 1969
Analysis of the predicted stDPE2 primary protein structure by the NCBI conserved domain search (Marchler-Bauer et al., 2002
An antibody was produced that recognizes specifically the presence of the protein in plants. It was raised against two peptides, both of which are present within the putative catalytic domain. To ensure the specificity of the antibody for stDPE2, it was affinity purified using the peptides. Immunoblot analysis indicated the presence of one protein of approximately 100 kD in molecular mass in both leaves and tubers of potato plants. A time course was performed to study how the amount of protein changed in leaf tissue during times of starch degradation. It is clear that in leaves, although the protein is present continuously, it increases in amount a just prior to sunset and reduces in amount just before sunrise (Fig. 1a ). It was also detected in tubers stored for 8 weeks either at room temperature or at 4°C. The amount of stDPE2 was greater in those stored at 4°C than those stored at room temperature (Fig. 1b).
These data would indicate that stDPE2 most probably has a role in starch degradation rather than starch synthesis. This is because the amount of stDPE2 increases during periods of starch degradation, in this case in leaves just before and after sunset and in tubers stored at 4°C. It is also present, however, at times of starch synthesis, such as in leaves during the light period, so based on these data a role in starch synthesis cannot be ruled out.
To study the role of stDPE2 in plant metabolism, an RNA interference (RNAi) construct was manufactured designed to repress the presence of the protein in potato. Thirty transgenic plants were screened by immunoblots, and three lines (8, 26, and 28) were identified that lacked the protein in their leaves (Fig. 1c). Two of these (8 and 28) were chosen for further study. It was also found that the protein was not present in tubers of the transgenic lines either when growing (Fig. 1d) or when stored for 8 weeks at 4°C (data not shown). To examine whether starch degradation was impaired in leaves of the transgenic lines, they were wrapped in aluminum foil for 24 h before being bleached with ethanol and stained with Lugols solution to visualize starch. It was found that leaves from both transgenic lines contained starch, in contrast to the control, which did not (data not shown). The amount of starch present in leaves at the beginning and end of the dark period was also determined for potato plants of three different ages (5, 10, and 15 weeks old; Fig. 2 ). The data are somewhat different between the differently aged plants, either because of changes in climate at the various sampling dates or because of the different ages of the plants. Nevertheless, for all ages the leaf starch contents of the plants were greater at the end of the night period in the transgenic plants in comparison with the control (Fig. 2). This indicates that stDPE2 is involved in starch degradation.
The Starch-Excess Phenotype Is Not Due to an Increased Rate of Starch Synthesis Although unlikely, it is still possible that the increase in starch seen in the leaves of the transgenic lines is because starch is synthesized faster there. To examine this, we kept plants without any light for 3 d to ensure that they contained no starch in their leaves. We then illuminated them and measured the amount of starch produced over a 10-h period. Plants from both the control and transgenic lines manufactured starch at a linear rate over this period, and no differences in amounts were found between the lines (data not shown). This demonstrates that the starch-excess phenotype found in the transgenic lines is caused by a lesion in the pathway of starch breakdown.
Potato tubers degrade starch and accumulate soluble sugars in their tubers when stored at low temperatures in a process known as cold sweetening (Müller-Thurgau, 1882
Analysis of an algal D-enzyme mutant indicated that the enzyme was involved directly in amylopectin biosynthesis (Colleoni et al., 1999a
To try and identify the possible substrate for the stDPE2 isoform, malto-oligosaccharides were isolated from the control and the transgenic plants. This is because analysis of an Arabidopsis dpe1 mutant demonstrated that it accumulated malto-oligosaccharides, especially malto-triose, which is its preferred substrate (Critchley et al., 2001
The malto-oligosaccharides within the samples were also analyzed by HPAEC-PAD to determine which accumulated in the transgenic lines (Fig. 3). It was found that maltose was present in both transgenic lines (Fig. 3, b and c) but was absent from the control (Fig. 3a). The amounts of no other malto-oligosaccharide were consistently altered in the transgenic lines. Maltose must be produced in plastids during starch breakdown, as it is the only product of  -amylase activity, and a -amylase isoform has recently been demonstrated to be involved in starch degradation in potato leaves (Scheidig et al., 2002). It has always been presumed that maltose would be degraded by -glucosidase isoforms; however, in the only study in which the activity of an -glucosidase was repressed in plants, it was demonstrated that it was most likely involved in processing glycoproteins (Taylor et al., 2000). This does not mean, of course, that no -glucosidases are involved in degrading maltose, but this remains to be proven.
It has been demonstrated recently in the dpe2 mutant of Arabidopsis that extracts made from these mutant plants lacked an activity catalyzing the transfer of glucose from maltose to glycogen, and it was assumed that this was the reaction catalyzed by atDPE2 (Chia et al., 2004 To quantify this activity, we incubated the purified protein with maltose and glycogen and measured glucose production enzymatically. We found that samples containing the protein released significant amounts of glucose, and this release was not detected when incubated with either maltose or glycogen alone. In addition, the amounts of glucose released increased linearly with time (data not shown). Based on this data, we calculated the specific activity of the protein, which is shown in Table II. We also tested whether the same activity could be found when we incubated stDPE2 with maltose and amylopectin. In this case, glucose was produced, but the activity was approximately 20-fold less than when glycogen was used as a substrate (Table II). As stDPE1 uses malto-oligosaccharides of many different degrees of polymerization as substrates, we decided also to test if stDPE2 could also metabolize malto-triose. When we incubated stDPE2 with malto-triose and glycogen, however, no glucose production was detected (Table II), indicating that stDPE2 uses exclusively maltose as a donor substrate.
To demonstrate that stDPE2 actually transfers glucose from maltose to glycogen, we repeated the experiment, but included [U-14C]maltose as a substrate. Following incubation, we precipitated the glycogen, washed it, and determined incorporation of radioactivity into it. It could be demonstrated that radioactivity became incorporated into the glycogen and that by using this data a similar specific activity for stDPE2 could be calculated, as was found when production of glucose was measured (Table II). This would be expected, as every transferase event should lead to the incorporation and liberation of equivalent amounts of glucose.
Glycogen itself may not be the acceptor substrate used by stDPE2 in the plant. Although glycogen is thought to be an intermediate of starch biosynthesis (Kossmann and Lloyd, 2000 These data also help to explain how stDPE2 was identified in the E. coli screening system that we used. E. coli manufacture glycogen, and stDPE2 could have used that as the acceptor substrate for maltose, allowing the production of glucose.
The activities of
The Protein Is Present in Potato Chloroplasts
Plants often contain multiple isoforms of starch degradative enzymes, some of which are extraplastidial. We decided, therefore, to examine the subcellular localization of stDPE2. To do this, we isolated chloroplasts from potato leaves using the method of Stitt et al. (1989)
Many of the data presented in this study in potato are extremely similar to those found in the studies of the Arabidopsis dpe2 mutants (Lu and Sharkey, 2004
Materials Escherichia coli strains were obtained from the CGSC E. coli genetic stock center. Unless otherwise stated, all chemicals were purchased from Sigma (St. Louis). Columns used for protein purification were purchased from Amersham Biosciences (Hillerød, Denmark).
A cDNA library was transformed in the E. coli strain TSM90 (CGSC no. 6145) and grown overnight at 37°C on MacConkey agar supplemented with 2% (w/v) maltose and the appropriate antibiotic. Plasmid DNA was isolated from 20 colonies demonstrating a maltose positive phenotype using a commercially available kit (Qiagen USA, Valencia, CA). These plasmids were named pMDE1 to pMDE20.
Sequencing of the inserts from the plasmids pMDE2, pMDE11, and pMDE18 was performed by a commercial company (AGOWA GmbH, Berlin).
An intron-less RNAi construct was produced in the plant transformation vector pBinARHyg by the following method. A SacI fragment of the cDNA pMDE11 was isolated from an agarose gel following electrophoresis, then blunted and ligated into the XhoI site of pMDE11, which also had been blunted. The orientation of the fragment was determined by restriction digests, and the new plasmid was named pMDE11RNAi. This plasmid was digested with the restriction enzyme XbaI, yielding a fragment of approximately 2,500 bp, which was ligated into the XbaI site of the binary vector pBinARHyg (Abel et al., 1996
Potato (Solanum tuberosum) L. cv Désirée was obtained from Saatzucht Fritz Lange KG (Bad Schwartau, Germany). Plants in tissue culture were kept under a 16-h-light/8-h-dark regime on Murashige and Skoog medium (Murashige and Skoog, 1962
Antibodies were manufactured by a commercial organization (Eurogentec, Seraing, Belgium) using their double XP protocol. In outline, rabbits were injected with two peptides present in the stDPE2 primary protein sequence (TGAPPDYFDKNGQNW and VRGSGRFYPQKDLESG). Following bleeding, immunoglobulins that recognize the peptides were affinity purified by the company.
Protein extracts were denatured by heating at 95°C for 5 min in sample buffer (Laemmli, 1970
Malto-oligosaccharides were isolated from leaves of potato plants by the method of Critchley et al. (2001)
Starch, sucrose, glucose, and fructose were determined according to the method of Müller-Röber et al. (1992)
For the determination of enzyme activities in plant material, frozen leaf discs were ground in extraction buffer containing 50 mM HEPES-KOH, pH 7.5, 5 mM MgCl2, 2 mM EDTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 8.7% (v/v) glycerol. The samples were centrifuged for 10 min at 4°C and 20,000g, and the supernatant was immediately frozen in liquid nitrogen before being stored at –80°C. The protein content of the supernatant was determined by the method of Bradford (1976)
Activities of starch degradative enzymes were measured in these samples.
Starch was isolated from tubers by the method of Lloyd et al. (1999)
Chloroplasts were isolated from leaves of 4-week-old potato plants by the method of Stitt et al. (1989)
Liquid chromatography was performed at 20°C using a ÄKTAexplorer 10 system from Amersham Biosciences. All other procedures were performed at 4°C. Columns used for D-enzyme purification were purchased from Amersham Biosciences.
Fifty grams of potato tubers were homogenized in 125 mL of ice-cold extraction buffer (50 mM HEPES-KOH, pH 7.5, 5 mM EDTA, 5 mM dithiothreitol, 5 mM MgCl2, 2 mM The following buffers were used in the enzyme purification: buffer A, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM 2-mercaptoethanol, and 1 M ammonium sulfate; buffer B, same as buffer A but without ammonium sulfate; and buffer C, same as buffer B, but containing 0.5 M KCl. The proteins were chromatographed by hydrophobic interactions using a RESOURCE ETH column (0.96-mL matrix) equilibrated in buffer A. After loading, a linear gradient (100%–0% in 25 column volumes [CV]) of buffer B was applied at a flow rate of 2 mL min–1. Fractions of 1 mL were collected and subsequently immuno-assayed for presence of stDPE2. Fractions containing stDPE2 were pooled and dialyzed against buffer B. The dialyzed protein was chromatographed on a RESOURCE Q (0.96 mL) column equilibrated in buffer B. The column was washed in 10 CV of buffer B before a linear gradient (0%–100% in 25 CV) of buffer C was applied at a flow rate of 2 mL min–1. Fractions of 1 mL were collected and immuno-assayed for presence of stDPE2. Fractions containing stDPE2 were pooled and dialyzed against buffer B. The dialyzed protein was chromatographed on a Mono Q (0.98 mL) column equilibrated in buffer B. The column was washed in 3 CV of buffer B before a linear gradient (0%–100% in 25 CV) of buffer C was applied at a flow rate of 0.5 mL min–1. Fractions of 0.5 mL were collected and immuno-assayed for presence of stDPE2. These fractions were pooled, aliquoted, and frozen at –80°C until use.
stDPE2 protein was incubated in 50 mM MOPS, pH 6.8, 30 mM maltose, and individual potential substrates. These were 5 mM malto-triose, maltoheptaose, maltopentaose maltohexaose, maltoheptaose, or 1.5% (w/v) oyster glycogen or potato amylopectin. Following incubation, the reactions were stopped by heating for 2 min at 90°C.
Three volumes of ethanol were added to the samples containing either glycogen or amylopectin, and the precipitated polysaccharides were removed by centrifugation. The sugars in 100-µL samples of all of these were separated by HPAEC, as described by Blennow et al. (1998)
stDPE2 was incubated as described above, although as a control maltose was omitted from some samples. As blanks, stDPE2 protein, which had been heated at 95°C for 5 min, was used. The amount of glucose in 50-µL samples was determined enzymatically by the method of Müller-Röber et al. (1992)
stDPE2 protein was incubated as described above with 1.5% (w/v) oyster glycogen and 92.5 MBq mol–1 [14C]maltose (Amersham). As blanks, stDPE2 protein, which had been heated at 95°C for 5 min, was used. Following incubation, glycogen was precipitated from 50-µL samples by the addition of three volumes of 75% (v/v) methanol and 1% (w/v) KCl. The precipitate was harvested by centrifugation at 20,000g for 10 min and then washed with 1 mL 75% (v/v) methanol and 1% (w/v) KCl, before being dissolved in 500 µL of water. Five milliliters of liquid scintillation fluid was added to this, and the amount of radioactivity determined. Activities were calculated as amount of radioactivity incorporated in the presence of stDPE2, subtracting the amount incorporated in the presence of the denatured protein. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY510449.
We thank Per Lassen Nielsen for assistance with the HPAEC-PAD and Romy Baran with the potato transformation. Received December 22, 2003; returned for revision January 20, 2004; accepted January 26, 2004.
1 This work was supported by PlantTec Biotechnology GmbH, Potsdam, Germany (grant to J.R.L.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.038026. * Corresponding author; e-mail james.richard.lloyd{at}risoe.dk; fax 45–4677–4109.
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ABSTRACT
RESULTS AND DISCUSSION
0.05) more malto-oligosaccharides than the control at both time points (
-aminocaproic acid, 0.5 mM phenylmethylsulfonyl fluoride, and 0.1% (w/v) Triton X-100). The slurry was filtered through two layers of nylon mesh and centrifuged at 2,000g for 10 min. Ammonium sulfate was added to the supernatant, and the protein fraction that precipitated between 25% to 50% (w/v) ammonium sulfate was isolated by centrifugation at 20,000g for 60 min. The precipitated proteins were dissolved in 50 mL of the sample buffer described above containing 1 M ammonium sulfate. 
