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Structural, Physicochemical, and Pasting Properties of Starches from Potato Plants with Repressed r1-Gene† Anders Viksø-Nielsen, Andreas Blennow, Kirsten Jørgensen, Karina H. Kristensen, Ann Jensen, and Birger Lindberg Møller* Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Received January 29, 2001; Revised Manuscript Received May 1, 2001
The aim of this work was to investigate the effect on starch molecular and physicochemical properties of down regulation of the R1 protein in potato (Solanum tuberosum L. cv. “Dianella”) tubers. Most prominent is a 90% suppression of the phosphate content in the isolated potato tuber starch. The amylopectin chain length distribution profile as determined by HPAEC/PAD was not affected, but the amylose content was increased in the most down-regulated plants. The pasting properties of the transgenic starch revealed a pronounced decrease in peak viscosity and increased setback viscosity as measured using a rapid Visco analyzer. The starch gels displayed an increased hardness and stickiness with a maximum at 1.7 nmol of Glc-6P mg-1 of starch compared to the control lines. At very low phosphate levels (1.4 nmol of Glc-6P mg-1 of starch), the gel hardness was decreased as a result of increased gel brittleness. The increase in gel brittleness is believed to be an effect of an increased proportion of free amylopectin blocklets in the starch as determined by SEC/RI. The possible links between the structural and physicochemical parameters are discussed. Introduction Starch is mainly composed of the two polymers amylose and amylopectin. Amylose is an essentially linear R(1f4)glucan molecule, whereas amylopectin is much larger and a highly R-1,6-branched molecule. The amylopectin molecules constitute approximately 75% of the starch granules and are packed as 400 nm semicrystalline blocklets.1 A fundamental property of amylopectin from tuberous plants, including potato, is that it contains relatively high amounts of covalently bound phosphate, i.e., one glucose unit out of 200300 is phosphorylated in potato.2 The phosphate groups are located as monoesters at the C-6 (approximately 70%) and at the C-3 (approximately 30%) positions of the glucose residues. Besides that, a small fraction (1%) may be linked to the C-2 position.2,3 Phosphorylation levels differ among potato varieties and is affected by growth conditions and P-fertilizer.4-6 The content of starch-bound phosphate is also affected by modulation of the starch branching enzyme and starch synthase activities.7,8 The observed correlation between the amylopectin unit chain length distribution profile and the degree of phosphorylation found in natural starches are thought to reflect variations in these enzyme activities.9,10 Phosphorylation proceeds concurrent with de novo synthesis of starch in potato tuber disks.11 Furthermore, it has been shown by Wischmann et al. that isolated potato amyloplasts * Corresponding author. E-mail:
[email protected]. Telephone: +45 35 28 33 52. Fax: +45 35 28 33 33. † Abbreviations: Glc-6P, glucose-6-phosphate; HPAEC/PAD, highpressure anion exchange chromatography with pulsed amperiometric detection; RVA, rapid Visco analyzer; SEC-RI, size exclusion chromatography-refractive index detection.
contain the full biosynthetic machinery for synthesizing phosphorylated starch using radiolabeled [33P]-Glc-6P as precursor.12 Recently, a protein designated R1, which partially is bound to the starch grain, has been isolated from potato.13 Antisense suppression of the R1 protein resulted in a 90% reduction of the content of starch-bound phosphate implying its direct or indirect involvement in potato starch phosphorylation.13 In addition the plants show reduced sensitivity to coldinduced sweetening. Expression of R1 in Escherichia coli increased the glycogen-bound phosphate more than 2-fold. The mechanism responsible for phosphorylation of starch and the role played by R1 in this respect remain elusive. The physicochemical properties, e.g., pasting and texture characteristics of a given starch sample are functions of its molecular constitution, e.g., amylose/amylopectin ratio, phosphate substitution, unit chain length distribution, and molecular size. For example, a high degree of phosphate substitution results in starch gels with high viscosity and stable starch pastes.14-16 For potato starch, the peak viscosity, as measured by a Brabender viscograph, is highly correlated to the degree of phosphate substitution as a possible direct effect of efficient hydration.17,18 However, the relationship between amylopectin chain length and phosphate substitution could partly restrict the hydration tendency of the highly phosphorylated starches.19 The enrichment of C-6 substituted phosphate in the amorphous regions of the starch granules suggests that increased hydration in amorphous starch is an important part of phosphate-mediated hydration during starch gelatinization.9 The rheological properties also greatly depend on the molecular size of the starch molecules.20,21 Recently,
10.1021/bm0155165 CCC: $20.00 © 2001 American Chemical Society Published on Web 06/22/2001
Properties of Starches from Potato Plants
aggregation between amylopectin molecules was demonstrated to exert a profound effect on starch viscosimetric properties.22 A significant quantity of industrial starch is chemically phosphorylated to improve its quality. Accordingly, it would be of great importance to control the level of starch-bound phosphate in vivo in order to enable production of new tailored starch-based biopolymers. However, detailed and exact structure/function relationships have been difficult to assess mainly as a consequence of lack of starch model systems where the effect of single specific alterations in molecular parameters can be compared. The use of transgenic starches offers a partial solution to this problem. But even in these systems, severe pleiotropic effects may complicate the interpretation of data. The suppression of both isoforms of branching enzyme in potato results in a higher phosphorylation degree and longer chains of the starch molecule.7 From a metabolic point of view, these effects are not understood. In this study, we present a coherent analysis of a number of parameters defining starch structure and correlate these to the obtained physicochemical properties of the starches. Transgenic potato lines in which the expression of the r1gene has been down-regulated by an antisense or cosuppression approach using the tuber specific patatin promoter constitute the experimental material. Materials and Methods Plant Material. Potato (Solanum tuberosum L. cv. “Dianella”) plants were grown in the greenhouse under a 16-h light (20 °C)/8-h dark (15 °C) photoperiod. Natural daylight was supplemented with artificial light of 140 W m-2. Generation of Transgenic Lines. Initially, the full-length R1 gene was cloned into pBlueScript.23 For generating the antisense construct, a Pst1 fragment was introduced in the reverse direction into the vector pDAN6 between the tuber specific patatin promoter and the CaMV 35S terminator.24 For the sense construct, an EcoRI/HindIII fragment was cloned directly into the pDAN8 vector between the patatin promoter and the CaMV 35S terminator. Both constructs were introduced into potato by Agrobacterium tumefaciensmediated transformation, and transgenic plantlets were selected on mannose-containing medium.25 Transforming potato stem cuttings with empty pDAN6 and pDAN8 vectors generated the corresponding control lines. Selection of Transgenic Lines. Initial screening of 45 antisense lines and 35 sense lines were performed by measuring the starch-bound Glc-6P content. Starch was extracted from all lines using a fruit-juicer.9 The amount of starch-bound Glc-6P was measured after acid hydrolysis of the starch.4 Five lines of each construct were selected as well as two control lines. Western Blot Analysis. To analyze the degree of R1 down-regulation, soluble and starch-bound proteins were extracted from the 12 selected lines using the method described by Denyer et al., and subsequently analyzed using SDS-PAGE (12-25% linear gradient gels).26 Proteins were electrotransferred to a nitro-cellulose membrane at 300 mA
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for 2 h using a semidry gel blotting apparatus. The membrane was blocked with 1% skim milk and then incubated overnight with a primary antibody (diluted 1:1000) directed toward R1. R1 bands were visualized using alkaline phosphatase conjugated secondary antibodies. Starch Analysis. The unit chain length distribution profile of the debranched starch was determined, using a DX 500 system (Dionex Corp., Sunnyvale, CA) equipped with an S-3500 autosampler and fitted with a CarboPac PA-100 column.9 The amount of amylose relative to the control was measured by iodine colorimetry using the method described by Bay-Smidt et al.27 Size Exclusion Chromatography and Refractive Index Detection (SEC/RI). Partial separation of amylose, amylopectin, and amylopectin aggregates was performed as described in detail elsewhere.22 Starch (10 mg) was wetted in 40 µL of 2 M NaOH, incubated for 15 min at room temperature, and transferred to a boiling water bath. The clear gel obtained was dissolved in 4.95 mL of H2O by gentle pipetting for 4 min and a 200 µL aliquot was applied to a Toyopearl TSK HW-75 F (Tosohaas, Cambridge, U.K.) column (diameter, 26 mm; height, 300 mm; exclusion limit, Mw ) 5 × 107) equilibrated with 10 mM NaOH and eluted with the same eluent (0.75 mL/min, 50 °C). Carbohydrate content was detected on-line by refractive index (Waters 410 Differential refractometer, Millipore, Bedford, MA). The maximum wavelength of the R-glucan-iodine complex was determined by mixing 900 µL aliquots of the SEC fractions with 100 µL of Lugol’s solution (0.2 g/L I2, 2 g/L KI, 1 M HCl) diluted 5 times. Spectra were recorded between 700 and 400 nm on a Shimadzu UV-160A spectrophotometer with automatic identification of λmax values. Pasting and Texture Characteristics. Starch pasting was monitored using a rapid Visco analyzer 4 (RVA, Newport Scientific, Warriewood, Australia) using 2.00 g of starch including 14% water, triplicate samples, and 25 mL of double de-ionized water. The starch suspension was stirred with a plastic paddle at 160 rpm in an aluminum container. The temperature was kept at 50 °C for 1 min, then heated to 95 °C at 12.3 °C/min, kept at 95 °C for 2.5 min, cooled to 50 °C at 12.3 °C/min, and finally kept at 50 °C for 2 min. The starch pastes obtained were kept sealed at room temperature for 15-24 h, and the resultant starch gels were subsequently subjected to texture analysis using a Texture analyzer (model TA-XT2i, Stable Micro Systems, Surrey, U.K.) fitted with a P4 flat probe. The analyzer was trigged at 0.010 N and the force measured at 0.9 mm/s for 7.5 mm. The time force profiles were evaluated using Texture Expert, version 1.22. The terms used to describe the texture are defined as follows: gel strength or hardness equals the positive area under the time force curve during the downward or compression whereas the stickiness or adhesiveness equals the negative area under the origin of the curve. Results Isolation of Transgenic Lines with Altered Starch Phosphate Content. Starch was isolated from potato tubers
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Table 1. Level of Starch-Bound Phosphate, Measured as nmol of Glc-6P mg-1 of Starch, in the 10 Selected Plant Lines with the Corresponding Control Lines construct
plant line
nmol of Glc-6-P mg-1 of starch
antisense
1 2 3 4 5 control 6 7 8 9 10 control
1.4 1.7 4.3 5.8 10.0 12.1 1.7 2.4 4.4 5.5 7.2 10.7
sense
Viksø-Nielsen et al. Table 2. Number of Tubers and Average Weight Per Tuber from R1 Antisense and Sense Potato Linesa plant lines no. of tubers av weight
antisense
sense
control
9.4 ( 0.9 11.9 ( 0.9
7.5 ( 0.5 15.4 ( 1.6
5.4 ( 0.3 23.0 ( 1.7
aThe values are means of all generated lines (140 antisense, 90 sense lines, and 20 control lines) ( SD.
Table 3. Amylose Content, Measured as Percentage of Total Starch, in R1 Antisense and Sense Plant Lines construct antisense
sense
plant line
nmol of Glc-6P mg-1 of starch
amylose content (%) ( SD
1 2 3 4 5 control 6 7 8 9 10 control
1.4 1.7 4.3 5.8 10.0 12.1 1.7 2.4 4.4 5.5 7.2 10.7
33.7 ( 0.1 32.8 ( 0.2 32.9 ( 0.1 29.3 ( 0.3 28.5 ( 0.1 28.7 ( 0.1 33.3 ( 0.2 28.4 ( 0.4 28.9 ( 0.2 28.1 ( 0.3 29.3 ( 0.2 28.9 ( 0.4
Figure 1. Western blot analysis of the degree of down-regulation of R1 in antisense and sense potato plants: (A) antisense lines; (B) sense lines. SBP: starch-bound protein. Soluble: soluble potato tuber protein. The numbers refer to plant lines and C indicates a control line. Protein extracted from equal amounts of starch was loaded in each lane in the SBP blots. Equal amount of protein was loaded in each lane in the blot of soluble protein.
from 45 antisense and 35 sense lines and analyzed for the amount of starch-bound phosphate as measured by nmol of Glc-6P mg-1 of starch. The phosphorylation level in the lines varied from 1.4 to 12.1 nmol of Glc-6P mg-1 of starch. The lowest degree of phosphorylation, 1.4 nmol of Glc-6P mg-1 of starch, was found in an antisense line. Five lines from each construct type, covering a wide range of phosphorylation levels, were chosen for further analysis (Table 1). Down-Regulation of R1. The R1 protein is partitioned between the starch granule and the stroma;28 hence, the level of R1 protein was analyzed using Western blotting of starchbound proteins (SBP) and soluble proteins from five antisense lines and five sense lines as well as from the corresponding control lines (Figure 1). In the most downregulated line, line 1, no R1 protein was detectable in the starch-bound fraction, whereas a small amount was found in the soluble fraction. A general trend that can be observed is that the lines with less than 5 nmol of Glc-6P mg-1 of starch has a very low content of R1, whereas there is no clear difference in R1 levels in the lines with more than 5 nmol of Glc-6P mg-1 of starch (lines 4, 5, 9, and 10). Thus, the decrease in starch-bound phosphate content is an effect of suppressed amounts of R1 protein, or at least in plant lines with less than 5 nmol of Glc-6P mg-1 of starch. Altered Phenotypes. The down-regulation of R1 resulted in minor phenotypical changes (Table 2). Mainly, the frequency of tuber initiation seems to have been affected. In the transgenic lines, the number of tubers was on average
Figure 2. Chain length distribution profile of the debranched starch samples from three selected lines as analysed by HPAEC: (b) line 1; (O) line 7; (1) control.
increased with 73% in the antisense plants and with 37% in the sense plants, whereas the average weight per tuber decreased with 48 and 32%, respectively, as compared to the control lines. The total yield from each plant, however, was on average the same in all lines, because increased tuber number was counteracted by a lower tuber weight, although a trend toward decrease in total starch yield could be noted. There were no differences in starch granule morphology and size as monitored by light microscopy (data not shown). Amylopectin Chain Length Distribution and Amylose Content. The chain length distribution profiles of the isolated starch samples were determined after debranching with isoamylase using Dionex chromatography. The profiles for three representative lines are shown in Figure 2. Only small changes among the profiles are observed, and the main peak in the three profiles emerges at the same DP. The amylose content in the starch granules relative to the control was also determined (Table 3). In the most downregulated lines (1, 2, 3, and 6) the amylose content was
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Properties of Starches from Potato Plants Table 4. Viscosimetric Data as Measured by RVA Analysis from Four Selected R1 Antisense and Sense Lines, Including One Control Line
plant line
nmol of Glc-6P mg-1 of starch
peak viscosity (cP)
peak time (min)
pasting temp (°C)
final viscosity (cP)
setback (cP)
1 2 6 7 control
1.4 1.7 1.7 2.4 12.1
2230 2590 2370 4950 7460
5.1 5.1 5.4 3.5 3.1
49.9 50.0 50.0 50.1 49.9
2490 2720 3350 2900 2770
870 770 1160 560 400
somewhat higher as compared to the two control lines, whereas in lines 4, 5, 7, 8, 9, and 10 containing more than 2 nmol of Glc-6P mg-1 of starch, the amylose content was identical to that of the control lines. Physicochemical Properties. Physicochemical properties of the transgenic starches including pasting, viscosity, and texture properties as well as molecular size/aggregation were investigated. The pasting properties of the isolated starches was measured using a rapid Visco analyzer (RVA) with a standardized protocol, and the resulting viscograms for three selected transgenic potato lines are presented in Figure 3. The most down-regulated line that contains 1.4 nmol of Glc6P mg-1 of starch showed a dramatically decrease in peak viscosity, 2230 vs 7460 cP in the control line (Table 4). The setback, which is defined as the difference between the minimum observed after the peak and the final viscosity, was increased more than 2-fold whereas the final viscosity was unchanged compared to the control. These effects were most prominent for the lines with less than 2 nmol of Glc6P mg-1 of starch (Table 4). The texture characteristics of the starch gels generated in the RVA measurements were further analyzed by texture analysis (Figure 4 and Table 5). The starches containing the least starch-bound phosphate exhibited pronounced textural changes. Most notably, the gels were more brittle and showed increased stickiness. The increased brittleness was monitored as a large shift in the time point at which the gel broke, i.e., at the maximal force. The gels with starches containing equal to or less than 1.7 nmol of G6P mg-1 of starch broke before the probe reached the lowest point at 8.3 min (7.5 mm). Another effect of the decreased starch phosphate content was a significant increase in gel-forming capacity (area I). However, the gel strength was suppressed below 1.7 nmol of G6P mg-1 of starch as a possible effect of increased brittleness. Maximum gel strength was thus found for one
Figure 3. Pasting properties of two selected R1 antisense and sense lines as determined using a rapid Visco analyzer: (s) line 1; (‚‚‚) line 7; (- - -) control. The temperature profile is indicated with a dotted line.
of the starches with 1.7 nmol of G6P mg-1 of starch (Table 5). Stickiness, as monitored by the adhesiveness of the starch paste to the probe (area III) was, however, highest for the two starches with the least starch phosphate content. The observed differences in the RVA and textural properties of the starches can possibly reflect alterations in the molecular size of the starch molecules. This was analyzed using SEC-RI (Figure 5). Using this system, three major constituents of starch, i.e., amylopectin large aggregates, free amylopectin blocklets, and amylose can be partially separated and analyzed.22 Amylopectin and amylose in the profile were identified using the λmax values of the iodine-R-glucan complex that is significantly higher for amylose than for amylopectin. As reported above, the starches with less than 2 nmol of Glc-6P mg-1 of starch (Figure 5A-C) were very different from those containing more than 2 nmol of Glc-6P mg-1 of starch (Figure 5D,E). The ratio between the amount of large amylopectin aggregates eluting at 66 mL and free 400 nm amylopectin blocklets eluting at 88 mL was severely decreased in the low phosphate lines. To investigate whether the observed aggregates were the result of noncovalent molecular interactions between amylopectin molecules (blocklets) or whether the aggregates were formed through establishment of stable, possibly covalent linkages between amylopectin blocklets, further analyses were performed at different starch concentrations (Figure 6). For the wild-type control line, the aggregate formation was positively correlated with the concentration of starch, indicating that at least part of the aggregates were formed as a result of noncovalent interactions, possibly through formation of double helical segments. For the low phosphate line (1.4 nmol of G6P mg-1 of starch) aggregate formation showed no concentration dependence, indicating
Table 5. Texture Analyses Data from Four Selected R1 Antisense and Sense Linesa plant line
nmol of Glc-6P mg-1 of starch
force (N)
area 1
area 2
area 3
1 2 6 7 control
1.4 1.7 1.7 2.4 12.1
0.052 ( 0.002 0.062 ( 0.003 0.087 ( 0.003 0.050 ( 0.001 0.032 ( 0.003
0.289 ( 0.005 0.339 ( 0.009 0.409 ( 0.000 0.209 ( 0.002 0.143 ( 0.003
0.014 ( 0.003 0.018 ( 0.001 0.030 ( 0.003 0.028 ( 0.000 0.018 ( 0.001
-0.032 ( 0.001 -0.028 ( 0.001 -0.021 ( 0.002 -0.004 ( 0.000 -0.014 ( 0.005
a The data are extracted from the texture analysis profiles presented in Figure 4. Values given are means of three independent measurements ( standard deviation.
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Figure 4. Texture analysis of stored starch samples, from RVA analysis: (A) line 1; (B) line 2; (C) line 6; (D) line 7; (E) control.
that the amylopectin blocklets are linked entirely by very stable, possibly covalent bonds. To further investigate this possibility, gelatinized starch samples were degraded with β-amylase, a hydrolytic exo-acting enzyme that by specific hydrolysis of R-1,4 bonds releases maltose units from the free nonreducing end of linear R-glucan chains in the starch molecule. The action of β-amylase is stopped by the presence of R-1,6 branches or phosphoester bonds. Because the wildtype control and line 1 have nearly identical chain length distribution profiles but line 1 has an 8-fold reduced phosphorylation level, starches from line 1 would be expected to be more prone to degradation by β-amylase compared to
Figure 5. SEC-RI of control and transgenic starches: (A) line 1; (B) line 2: (C) line 6; (D) line 7; (E) control. On the graphs is indicated amount of Glc-6P mg-1 of starch and the approximate molecular weights and radii of gyration of amylopectin aggregates, amylopectin blocklets, and amylose as given in Blennow et al.22
wild-type starch. Presence of continuous covalent bonds between individual amylopectin blocklets would reduce the sensitivity toward β-amylase, and the amylopectin molecular size would be less decreased. If, on the other hand, the blocklets are held together by loose R-glucan interactions,
Properties of Starches from Potato Plants
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Figure 6. Concentration dependent amylopectin aggregation and β-amylase susceptibility of gelatinized starch as analysed by SEC-RI: left, control line; right, antisense line 1. The amount of gelatinized starch applied to the column is indicated.
the resulting product would be of significantly lower molecular size, i.e., somewhat smaller than an amylopectin blocklet. Both the wild-type control starch and starch isolated from the low phosphate line were degraded to some extent as observed by an overall shift toward larger elution volumes. However, starch from line 1 was markedly more resistant to β-amylase treatment than the control starch (Figure 6). These results demonstrate the presence, both in the wild-type control line and in line 1, of covalent linkages between the 400 nm amylopectin blocklets, but these covalent linkages are more prominent in the transgenic starch. Discussion The effect of down-regulating of the starch-bound R1 protein on the physiology of the whole plant and to less
extent on starch properties has previously been investigated by Lorberth et al.13 In this study, we present a more thorough starch analysis on plants with down-regulated R1 protein (Figure 1) using a tuber specific promoter. A series of potato lines with gradually reduced R1 levels in their tubers were obtained. The maximal reduction in starch-bound phosphate as a result of the down-regulation of R1 corresponded to 10% of wild-type phosphate levels (Table 1) as also observed using antisense lines generated using the constitutive CaMV 35S promoter.13 Western blot analysis showed that the content of the R1 protein was clearly reduced in lines having less than 2 nmol of Glc-6P mg-1 of starch. Surprisingly, no apparent downregulation of the R1 protein was observed in the lines with more than 2 nmol of Glc-6P mg-1 of starch, although some of these lines had a significantly reduced starch phosphate
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content. Such a pattern would be expected if the R1 protein exerts its effect on starch phosphorylation as a regulator where small changes around a threshold value may have a large effect on the final phosphorylation level achieved. These observations correspond with those of Ritte et al.; i.e., there is no clear correlation between R1 content and the degree of phosphorylation, at least at levels above 2 nmol of Glc-6P mg-1 of starch.28 The pasting properties of the starches (Figure 3, Table 4) revealed a pattern similar to that found by Lorberth et al.; i.e., the peak viscosity was dramatically suppressed while the setback was increased.13 The decreased peak viscosity is most probably a direct effect of reduced starch granule hydration due to the low phosphate content.17,18 The potato lines with less than 2 nmol of Glc-6P mg-1 of starch had somewhat higher amylose content as compared relative to the control lines but the amylopectin chain length distribution analysis of all lines did not reveal any significant differences in the amylopectin fine structure that could account for the observed changes in pasting behavior. The higher amylose content may restrict starch granule hydration and swelling somewhat, due to rapid entanglement of the linear molecules and also explain the increased setback in line 1 since amylose free potato starch has a severe decrease in final viscosity.29,30 However, in the lines with virtually no changes in amylose content (e.g., line 7) significant changes in pasting properties was observed, demonstrating the pure effects of starch-bound phosphate on pasting characteristics including decreased peak viscosity and somewhat increased setback. The textural effects of suppressed starch phosphate content, i.e., the increased gel-forming capacity, brittleness, and stickiness may also be direct functions of the abovementioned structural parameters. The enhanced gel-forming capacity is probably directly linked to increased double helical network formation due to suppressed hydration capacity of the starch paste. However, at very low phosphate levels, the gels were more turbid and fragile, and as a result, the breakpoint of the gel appeared at much lower forces. This property could be a combined effect of the increased proportion of low molecular size amylopectin particles as determined by SEC-RI and severely depressed hydration capacity. The increased stickiness could likewise be a function of increased molecular surface made available by the larger proportion of low molecular amylopectin blocklets. The reduced capability of β-amylase to attack and subsequently trim the gelatinized starch from line 1 indicates the presence of a high proportion of covalent linkages between the amylopectin blocklets that effectively reduces the proportion of free nonreducing ends involved in intermolecular glucan double helix formation. We hypothesize that the altered structural properties and reduced susceptibility of the transgenic starches to β-amylase as described in this work is directly linked to the observed repressed cold-induced sweetening (starch excess phenotype) in R1 antisense potatoes.13 This work demonstrates a central role of the R1 protein in the primary metabolism of potato. Its absence results in severely reduced starch-bound phosphate content, increased amylose content, increased ratio of free amylopectin block-
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lets, which furthermore are covalently linked by continuous glucosidic bonds. No single metabolic reaction can at present be assessed to explain such diverse and seemingly unrelated effects, in particular, the links between starch-bound phosphate and degradation, degree of branching, amylose content, and amylopectin blocklet linkage as demonstrated in this work.7,13,28 Acknowledgment. We gratefully acknowledge Peter Poulsen, Danisco-Cultor Innovation, for the gift of the transformation vectors; Hanne Engel, Danisco-Cultor Innovation, for excellent technical assistance in the transformation and regeneration of potato plants; and Håkan Larsson and Patrick Hao-Jie Chen, Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden for the initial gift of the R1 cDNA clone. This work was financially supported by the Danish National Research Foundation and by a nonfood grant from the Danish Agency for Trade and Industry. References and Notes (1) Atkin, N. J.; Abeysekera, R. M.; Cheng, S. L.; Robards, A. W. Carbohydr. Polym. 1998, 36, 193. (2) Hizukuri, S.; Tabata, S.; Nikuni, Z. Starch/Sta¨ rke 1970, 22, 338. (3) Takeda, Y.; Hizukuri, S. Carbohydr. Res. 1982, 102, 321. (4) Bay-Smidt A. M.; Wischmann, B.; Olsen, C. E.; Nielsen, T. H. Starch/Sta¨ rke 1994, 46, 167. (5) Nikuni, Z.; Hizukuri, S.; Kamagi, K.; Hasegava, H.; Moriwaki, T.; Fukui, T.; Doi, K.; Nara, S.; Maeda, I. Mem. Sci. Indstr. Res. Osaka UniV. 1969, 26, 1. (6) Jacobsen H. B.; Madsen, M. H.; Christiansen, J.; Nielsen, T. H. Potato Res. 1998, 41, 109. (7) Schwall, G. P.; Safford, R.; Westcott, R. J.; Jeffcoat, R.; Tayal, A.; Shi, Y.-C.; Gidley, M. J.; Jobling, S. A. Nature Biotechnol. 2000, 18, 551. (8) Abel, G. J. W.; Springer, F.; Willmitzer, L.; Kossmann, J. Plant J. 1996, 10, 981. (9) Blennow, A.; Bay-Smidt, A. M.; Wischmann, B.; Olsen, C. E.; Møller, B. L. Carbohydr. Res. 1998, 307, 45. (10) Blennow, A.; Engelsen, S. B.; Munck, L.; Møller B. L. Carbohydr. Polym. 2000, 41, 163. (11) Nielsen, T. H.; Wischmann, B.; Enevoldsen, K.; Møller B. L. Plant Physiol. 1994, 105, 111. (12) Wischmann, B.; Nielsen, T. H.; Møller, B. L. Plant Physiol. 1999, 119, 455. (13) Lorberth, R.; Ritte, G.; Willmitzer, L.; Kossmann, J. Nature Biotechnol. 1998, 16, 473. (14) Yamada, T.; Morimoto, Y.; Hisamatsu, M.; Komiya, T. Starch/Sta¨ rke 1987, 39, 208. (15) Davies, L. Food Technol. Eur. 1995, June/July, 44. (16) Muhrbeck, P.; Svensson, E.; Eliasson, A.-C. Starch/Sta¨ rke 1991, 43, 466. (17) Veselovsky, I. A. Am. Potato J. 1940, 49, 330. (18) Wiesenborn, D. P.; Orr, P. H.; Casper, H. H.; Tacke, B. K. J. Food. Sci. 1994, 59, 644. (19) Blennow, A.; Bay-Smidt, A. M.; Olsen, C. A.; Møller, B. L. Int. J. Biol. Macromol. 2000, 27, 211. (20) Willet, J. L.; Millard, M. M.; Jasberg, B. K. Polymer 1997, 38, 5983. (21) Sriburi, P.; Hill, S. E.; Barclay, F. Carbohydr. Polym. 1999, 38, 211. (22) Blennow, A.; Bay-Smidt, A. M.; Bauer, R. Int. J. Biol. Macromol. 2001, 28, 409. (23) Viksø-Nielsen, A.; Larsson, H.; Chen P. H.-J.; Blennow, A.; Møller, B. L. Submitted for publication in 2001. (24) Liu, X. Y.; Rochasosa, M.; Hummel, S.; Willmitzer, L.; Frommer, W. B. Plant Mol. Biol. 1991, 17, 1139. (25) Bojsen, K.; Donaldson, I; Haldrup, A.; Joersboe, M.; Kreiberg, J. D.; Nielsen, J.; Okkels, F. T.; Petersen, S. G. Patent Application, International Application Number PCT/EP94/00575, International Publication Number WO 94/20627. 1994.
Properties of Starches from Potato Plants (26) Denyer, K.; Hylton, C. M.; Jenner, C. F.; Smith, A. M. Planta 1995, 196, 256. (27) Bay-Smidt, A. M.; Blennow, A.; Bojko, M.; Møller, B. L. Ann. Trans. Nordic Rheol. Soc. 1999, 7, 31. (28) Ritte, G.; Eckermann, N.; Haebel, S.; Lorberth, R.; Steup, M. Starch/ Sta¨ rke 2000, 52, 145.
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