Phosphofructokinase in Relation to Sugar Accumulation in Cold-Stored Potato Tubers

J.PlantPhysiol. Vol. 138.pp. 129-135 (1991)

Phosphofructokinase in Relation to Sugar Accumulation in Cold-Stored Potato Tubers G. M. M. BREDEMEIJER', H. c.].

BURG

2

,

P. A. M.

CLAASSEN

3

,

and W.].

STIEKEMA

Centre for Plant Breeding Research CPO, P.O. Box 16, 6700 AA Wageningen, The Netherlands I Present address: Centre for Variety Research and Seed Technology CRZ, P,O. Box 32, 6700 AA Wageningen, The Netherlands 2 Present address: Royal Sluis, P.O. Box 22, 1600 AA Enkhuizen, The Netherlands 3 Research Institute ATO, P.O. Box 17,6700 AA Wageningen, The Netherlands Received October 11, 1990 . Accepted February 2,1991

Summary The accumulation of sucrose and reducing sugars in potato tubers (Solanum tuberosum L. cv. Bintje and a breeding clone KW74-2618) during storage at 2 ec occurs with a concomitant decrease in maximum activity of the assumed cytosolic phosphofructokinase (PFK). This decrease is most extensive in cv. Bintje. The change is reversed when the tubers are transferred to 13 ec. The activity of plastidic PFK hardly changes during the same experiments. The Arrhenius plots of cytosolic and plastid PFK are biphasic, showing a discontinuity at about 12.5 ec and 14.5 ec, respectively, with an increased slope as temperature is reduced to 2 ec, indicating cold sensitivity. However, no cold inactivation of the partially purified PFK enzymes has been observed during incubation at low temperature during 90 min. The results are discussed in relation to the view that during cold storage of potato tubers diminished PFK activity plays a role in the reduction of the rate of glycolysis, causing sugars to accumulate.

Key words: Solanum tuberosum, A TP dependent phosphofructokinase, cold-induced sweetening, temperature coefficient. Abbreviations: PFK = phospofructokinase (EC 2.7.1.11); PFK 1 and PFK 2 forms of PFK; QIO = temperature coefficient.

Introduction The accumulation of sucrose and reducing sugars in potato tubers stored at low temperatures has been suggested to depend on more than one cause (Pollock and ap Rees, 1975). In the first place modifications of the amyloplast membranes at low temperatures may lead to increased permeability and thus to the release of degradative enzymes and intermediates (Ohad et al., 1971; Workman et al., 1976; Isherwood, 1976). Secondly, the cold-induced increase in invertase activity may stimulate the formation of reducing sugars from sucrose (Pressey and Shaw, 1966). Finally, accumulation of sucrose and reducing sugars may result from a reduction in glycolysis as suggested by Dixon and ap Rees (1980 a), who © 1991 by Gustav Fischer Verlag, Stuttgart

=

two molecular weight

showed an accumulation of hexose phosphates and a subsequent increase in sucrose concentration following exposure of potato tubers to low temperatures. This block is presumably caused by cold lability of certain glycolytic enzymes (Pollock and ap Rees, 1975; Dixon et al., 1981). No coarse control of glycolytic enzymes was observed, including the two cold labile enzymes glyceraldehyde phosphate dehydrogenase (Pollock and ap Rees, 1975) and phosphofructokinase (Hammond et al., 1990). In this investigation, a time course study of phosphofructokinase (PFK) activity during storage of potato tubers for 6 months at 2 and 8 ec was undertaken. We used the Dutch commercial potato cultivar Bintje, which exhibits cold-induced sweetening (van Es and Hartmans, 1987) and

130

G. M. M. BREDEMEIJER, H. C. J. BURG, P. A. M. CLAASSEN, and W. J. STIEKEMA

a new breeding clone, KW74-2618, which is less cold sensitive. It was the purpose of this study to determine whether the activity of PFK, a key enzyme in glycolysis, catalyzing the A TP dependent conversion of fructose 6-phosphate to fructose 1,6-biphosphate, changes in a way that is consistent with the changes in sugar concentrations. Furthermore, we present additional information on the subcellular localization of different molecular weight forms of PFK in potato tubers to support the interpretation of changes in activity during cold storage. This was done as the majority of plants have a cytosolic PFK and a plastid PFK that differ in molecular and kinetic properties (Kelly and Latzko, 1977; Garland and Dennis, 1980; Hausler et al., 1987; Botha et al., 1988).

Materials and Methods Materials Tubers of Solanum tuberosum L. cv. Bintje and of a new genotype, KW74-2618 were grown in a randomized field design. This new clone is diploid and selected from multiple crosses between Solanum tuberosum cv. Sirtema and various Solanaceae obtained from the Commonwealth Potato Collection. The ancestry of KW74-2618 is: S. goniocalyx x ~ [(So chacoense x S. phureja) x Sirtema] (Colon et al., 1989). After harvest, tubers were kept for 2 weeks at 17°C for suberization and subsequently stored at 2 or 8°C in thermostatically controlled and ventilated cells in the dark. Throughout the storage experiment tubers were collected and used for the extraction of sugars. PFK activity was always measured in extracts prepared from the same potatoes. Biochemicals and lyophilized (sulphate-free) coupling enzymes for PFK assays were obtained from Sigma except for NADH, which was obtained from Boehringer, Mannheim. The Sephadex G-25 columns were from Pharmacia and the reagents for the protein assay from Biorad. Preparation of extracts For preparation of tuber extracts the procedure described by Dixon et al. (1981) was slightly modified. A representative tissue sample of 5 peeled tubers (120 g fr wt) was homogenized in a Waring Blendor for 1 min in 260 mL 50 mM Tris-HCI, pH 8.0, containing 2 mM EDTA, 5 mM dithiotreitol, 0.1 % (w/v) bovine serum albumin and 5 % (w/v) Dowex-1 resin (chloride form). The homogenate was filtered under reduced pressure and centrifuged at 35,000 x g for 30 min. An aliquot of the supernatant was used for the assay of total PFK activity and the remainder was used for gel filtration. Recovery of PFK activity during extraction was determined by adding known amounts of previously purified enzyme activity to the extraction buffer. The recovery experiments were carried out with control tubers and tubers that had been stored at 2°C for different time intervals. PFK from potato leaves was extracted similarly as described for tubers using 5 mL extraction buffer per g fresh weight. Isolation of chloroplasts and preparation of chloroplast extracts was according to Cseke et al. (1982). All procedures were carried out at 4°C. Gel filtration chromatography Solid ammonium sulphate was added to the crude extracts to 60% (w/v) saturation, and after equilibration for 1 h the suspen-

sion was centrifuged for 15 min at 10,000 x g. The pellet was resuspended in 20 mM imidazole, 80 mM KCI buffer, pH 6.5, and dialyzed for 2 h against 300 volumes of this buffer. The concentrated extract was applied to a Sephacryl S-300 column (2.5 x 90 cm) and eluted with buffer at a flow rate of 0.5 mL . min - 1. For molecular weight determination the Sephacryl S-300 column and the Superose 6 column were calibrated with blue dextran (2 MDa), thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa), ovoalbumin (43 kDa), chymotrypsinogen (25 kDa) and ribonuclease (13.7 kDa) as standards. Enzyme assay Enzyme activity was determined in crude extracts after Sephadex G-25 chromatography to remove low molecular weight metabolites. The maximum PFK activity was measured spectrophotometrically at 25°C by following the oxidation of NADH at 340 nm. The assay mixture described by Dixon et al. (1981) has been adapted and contained 4 mM fructose 6-phosphate, 2.6 mM ATP, 8 mM MgCb, 0.1 mM NADH, 4 mM cysteine, 1 unit aldolase, 1 unit aglycerophosphate dehydrogenase and 10 units triose phosphate isomerase in 40 mM Tris-HCI, pH 8.0, in a total volume of 3 mL. The reaction was started by the addition of PFK. Rates of NADH oxidation in the absence of fructose 6-phosphate or ATP were substracted from the total activity. Temperature coefficients were calculated from measurements made in the range of 2 to 25°C using the standard assay mixture. In a number of experiments Tris-HCI buffer was replaced by Hepes-KOH buffer to exclude the effect of pH shifts due to a decrease in temperature (Angelopoulos and Gavalas, 1988). Results in both buffers were similar. Protein concentration was determined as described by Bradford (1976) using bovine serum albumin as a standard. Analysis of sugars Sugar concentration was determined in lyophilized tuber tissue. After pulverization, sugars were extracted using 2.5 g powder in 80 % (v/v) boiling aq. methanol. The suspension was filtered and methanol was removed by evaporation under reduced pressure. The residue was resolved in 100 mL demineralized water. Hexose and sucrose concentrations were measured enzymatically in separate samples of 50-100 JtL according to the instructions of the Boehringer assays for determination of glucose, fructose and sucrose (see also Bergmeyer et aI., 1974).

Results Sugar contents in tubers Sugar contents of Bintje and KW74-2618 tubers during storage at 2 °C or 8 °C from a typical experiment are shown in Fig. 1. The concentration of reducing sugars remained nearly constant during the 1sl week of storage at 2 °C and then increased considerably. The rate and extent of the increase in Bintje exceeded that in KW74-2618. In KW74-2618 the level of reducing sugars reached a maximum after about 8 weeks and then decreased slightly. On the contrary, in Bintje the increase continued until after 8 weeks, albeit at a slower rate. The sucrose content in both genotypes started to increase already within 1 week and reached a maximum level prior to the reducing sugars. Storage at 8°C had little effect on the sugar concentrations in Bintje and KW74-2618 tubers (Fig. 1). Similar results were obtained in 2 other experiments

131

PFK in cold stored potato tubers 5.0

2.0

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4.0

0.4

3.0

0.3

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1.0

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--_-----_--------:::-:::-~-"'"KW -B°C

---=~====::======:::::::::::::::-------- .. Bi

o

50

100

150

-8 °C

0.4

4.0

PFK 1

200

Days in storage

Fig. 1: Changes in sugar content of Bintje (e) and KW74-2618 (_) potato tubers during storage at 2 °C (--) and 8°C (- - - - -).

in previous years. Cold-stored potato tubers of Bintje always accumulated reducing sugars and sucrose to an average concentration of 1.81±0.32 % and 0.64±0.05 % (fresh weight) over 3 years respectively. In KW74-2618 always a peak in the concentration of the sugars was observed amounting to an average maximum of 1.19 ± 0.18 % for reducing sugars and to 1.04±0.24% for sucrose. PFK in control tubers

Maximum PFK activities of suberized control tubers were 80.2±4.5 and 93.6±10.4mU per g fresh wt (mean±SE; n = 4) for Bintje and KW74-2618, respectively. Expressed on a protein basis the activities are 14.6±0.8 and 18.0±2.0 mU per mg protein for Bintje and KW74-2618. The values are comparable to those reported by Kruger et al. (1988) for tubers of cv. Record. PFK activity from both Bintje and KW74-2618 control tubers was resolved into two peaks by gel filtration on Sephacryl S-300 (Fig. 2). The first peak (PFKl) eluted just after the void volume of the column and corresponded to a molecular weight of 950 ± 85 kDa; the molecular weight of the second peak (PFK2) was 173 ± 5kDa. The percentage of PFKl of total PFK activity was 32 and 37 in Bintje and KW74-2618, respectively. In two experiments, average recovery of added PFKl was 85 % in Bintje and 88 % in KW742618. The recovery of PFK2 in five experiments was 82±4 % for Bintje and 78±4% for KW74-2618. The difference in PFKl activity between Bintje and KW74-2618 was neither due to differences in extractability nor to differences in stability during gel filtration (recovery of PFK from Sephacryl S-300 column in both cases about 85 %). The stability of PFKl and PFK2 was optimal at pH 6.5 for both genotypes. However, at this pH half of the activity of PFKl

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Fraction number

Fig. 2: Sephacryl 5-300 gel filtration of PFK from control tubers of Bintje (A) and KW74-2618 (B).

disappeared within 24 h, but the decrease in PFK2 was less than 5%. PFK in tubers during storage at 2°C or 8 °C

The changes in total PFK activity expressed in units per g fresh weight were relatively small during 6 months storage at 2°C. Total PFK activity decreased about 10% during the 1st week and then remained fairly constant during 4 months both in Bintje and KW74-2618. Some selected extracts were tested with respect to the recovery of PFK activity. In 2 experiments average recovery of PFK in extracts of Bintje was 87 % in controls and 84 % after 4 weeks of storage at 2 dc. For KW74-2618 values were 78 % and 71 %, respectively. When distinguishing the two molecular weight forms, a pronounced decrease in PFKl activity was observed in Bintje (Fig. 3 a). PFKl activity decreased rapidly within the 1st week at 2°C, thereafter the activity declined gradually. After reaching a minimum that amounted to about 50 % of the original activity, PFKl activity remained fairly constant during the course of the experiment. The decrease in PFKl activity in KW74-2618 during storage at 2°C was much smaller as compared with Bintje (Fig. 3 a). Changes in PFK2 were relatively small in both genotypes. No sprouting occurred throughout storage at this temperature.

G. M. M. BREDEMEIJER, H. C. J. BURG, P. A. M. CLAASSEN, and W. J. STIEKEMA

132

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130 120

Days reconditioned

- ..... Kw-BOC

30 20 10 00

Table 1: Changes in PFK activity and sugar content of potato tubers after storage for 20 weeks at 2 °C and subseq4ent transfer frojTI 2 °C to 13 0c.

Bintje

50

150

100

B PFK2

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150

0 3 9 16 22 KW74-2618 0 24 n.d. = not determined

PFK activity (mU/g fr.wt.) PFK1 PFK2 9.7 14.6 16.0 18.4 17.0 22.9 28.0

43.7 47.2 43.3 45.6 49.1 49.0 54.2

sugar content

(g/100 g fr.wt.)

sucrose 0.44 0.27 n.d. 0.16 0.11 0.79 0.14

reducing sugars 1.65 2.14 n.d. 1.39 0.93 0.90 0.05

200

Days in storage

Fig. 3: Changes in PFK1 and PFK2 in Bintje (e) and KW74-2618 (.) tubers stored at 2°C (--) or 8°C (- - - - -). Initial levels in mU/g fr.wt are for Bintje PFK1: 21.7; PFK2: 45.8 and for KW742618 PFK1: 34.3; PFK2: 59.3.

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When tubers were stored at non-sweetening conditions, at 8°C, PFK1 activity also decreased. In contrast to the decline in activity at 2 °C, this decrease occurred at a slower rate and continued for a longer period of time (Fig. 3 a). The alterations in PFK2 activity were again relatively small (Fig. 3 b). In tubers stored for 8 weeks at 17°C following harvest, the changes in activities of PFK1 and PFK2 were similar to those in tubers kept at 8 dc. At both temperatures tubers of Bintje and KW74-2618 showed sprouting after storage during 5 weeks. The data from similar storage experiments carried out in 2 previous years also showed the initial decrease in PFK1 activity and the higher PFKl activity in KW74-2618 as compared to Bintje after prolonged storage at 2°C (results not shown).

Reconditioning oj tubers In a first experiment Bintje and KW74-2618 tubers were transferred to 13 °C after 20 weeks storage at 2 °C and analysed after 3 weeks for activities of PFK1 and PFK2. During that period, PFK1 activity increased with 133 % and 23 % in Bintje and KW74-2618, respectively, while increases in PFK2 activity were relatively small, i.e. 14 % in Bintje and 12 % in KW74-2618. In a second experiment a time course study of the changes in activity of PFK and, in addition of sugars in Bintje was made. After transfer of the tubers from 2 °C to 13 °C sucrose and after some delay, the reducing sugars decreased with time. An increase in PFK1 activity accompanied the changes in sugar content (Table 1). In the case of KW74-2618, this so-called reconditioning also resulted in a decrease in sugars and an increase in PFK1 (Table 1). In both cases the initial level of PFK1 was almost attained. When tubers were kept at 2°C PFK1 activities did not change.

< .3>

Cl

0.5

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33

34

35

36

37

(1fT OK X104)

Fig.4: Arrhenius plots for PFK1 (.--.) and PFK2 (e--e) from control tubers of KW74-2618.

Again changes in PFK2 activity were relatively small in both genotypes (Table 1).

Effect oJlow temperature on PFK in vitro In order to study the effect of low temperature on PFK activity, the approach described by Angelopoulos and Gavalas (1988) was followed. First, the activity of the enzyme was determined at different temperatures over a range from 2 to 25°C and second the effect of incubation at low temperature on the maximum catalytic activity, assayed at 25 DC, was investigated. PFK assays using enzyme obtained from KW74-2618 performed at temperatures from 2 to 25°C revealed that the Arrhenius plots for both PFK1 and PFK2 were biphasic, showing a discontinuity at 14.4 ± 1.0 °C and 12.3 ± 1.2 DC, respectively, with an increased slope as temperatures were reduced to below 12 °C (Fig. 4). Similar results were obtained with Bintje. In this cultivar the discontinuity in the Arrhenius plot of PFK1 was at 14.9± 1.6 °C and that of PFK2 at 12.5 ± 0.0 dc. Temperature coefficients calculated

PFK in cold stored potato tubers Table 2: Temperature coefficients of PFKl and PFK2 from Bintje and KW74-2618 tubers.

A Leal

1.8

Temperature coefficient* 2-9°C

PFK2

1.6

15-25°C

enzyme

Bintje

KW74-2618

Bintje

KW74-2618

1.4

PFK1 PFK2

3.01±0.10 3.77±0.15

3.44±0.14 3.97±0.59

1.82±0.02 1.96±0.04

2.01±0.03 2.08±0.05

1.2

* Values are means±s.e. of four independent experiments

1.0

sulphate

Concentration Tuber Leaf Chloroplast mM PFK1 PFK2 PFK1 PFK2 PFK2 8 25 33 10 20 50

+11 +13 + 11 + 6 + 6 -11

-10 -19 -26 0 -10 -39

n.d. +9 n.d. n.d. +5 n.d.

-14 -23 -32 - 6 -23 -41

n.d. -34 n.d. n.d. n.d. -43

n.d. = not determined Values are means of two independent experiments

0.2

0.6

Table 3: Effect of phosphate and sulphate on the activities of PFK1 and PFK2 from KW74-2618 tubers and leaves and PFK2 in the chloroplast fraction. Increase (+) or decrease (-) activity in percentage of control.

phosphate

0.4

0.3

0.8

Effector

133

0.4 PFK1

~

0.2

iil N «

""'~ ~

o .............................. ................ 5

10 15 20 25 30 35 40 45

Localization of PFKl and PFK2 The study of localization of the different forms of tuber PFK was indirectly approached by isolating PFK activity from potato leaf chloroplasts. After chromatography of an extract of KW74-2618 leaves on 5ephacryl 5-300, the resolution of PFK was similar to that observed for tuber PFK (Fig. 2 b). Chromatography of leaf or tuber extracts on a FPLC-5uperose 6 column also revealed two PFK peaks. Leaf PFK eluted in a broad peak, probably a mixture of aggregates with molecular weights ranging from about 669 kDa to at

0

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PFK2

1.4

B Chloroplast 0.3

1.2

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0.6

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from these plots are summarized in Table 2. In all cases, the QlO values at 2 - 9°C were higher than at 15 - 25°C, indicating that in both genotypes PFKl and PFK2 are cold sensitive. In crude extracts of Bintje and KW74-2618 prepared at 4 °C the yield of total PFK was about the same compared with extracts prepared at 20°C; incubation of these extracts, undiluted or tenfold diluted, at respectively 4 and 20 °C at pH 8 during 2 h did not affect enzyme activity. Furthermore, when PFKl and PFK2 were incubated separately at pH 8, the pH at which cold sensitivity was established, again no significant differences were obtained between 4 and 20°C. Thus, incubation of the PFK's at 4°C did not show the characteristics of cold labile enzymes of which the activity is lost faster and to a greater extent at lower temperatures (Bock and Frieden, 1974). It is not excluded however, that by rapid reactivation during the higher temperature used for the assay of PFK, cold inactivation is masked.

0.1

0.1

£~

0.2

0

o ................................................ . 5

10

15

20 25 30 35 40 45

Fraction number

Fig. 5: FPLC Superose 6 profiles of potato leaf PFK (A) and potato chloroplast (B) of KW74-2618.

least 5 million Da and a sharp peak of about 180 kDa (Fig. 5 a). The broad and the sharp peak corresponded to PFKl and PFK2 from 5ephacryl 5-300 as established by rechromatography. Chromatography of an extract of isolated chloroplasts showed only one major peak corresponding to the sharp peak of leaf extracts (Fig. 5 b), indicating that only PFK2 is present in chloroplasts and that PFKl is located in the cytosol. However, these results do not rule out the possibility that PFK2 also occurs in the cytoplasm. Because of the often observed differential response of cytosolic and plastid types of plant PFK to anions we investigated the effect of phosphate and sulphate ions on the two PFK forms from tubers and leaves of KW74-2618. At pH 8.0, tuber PFKl was slightly stimulated by phosphate concentrations up to 33 mM and sulphate concentrations up to 20 mM (Table 3). In contrast, tuber PFK2 was progressively inhibited by increasing concentrations of these anions. The effects of the anions on PFKl and PFK2 from leaves were similar to those observed for the corresponding forms from tubers. The PFK2 in the chloroplast fraction was inhibited by both anions (Table 3).

134

G. M. M.

BREDEMEIJER,

H. C. J.

BURG,

P. A. M.

CLAASSEN,

Discussion The occurrence of two molecular weight forms of PFK observed in Bintje and KW74-2618 (Fig. 2) is supported by findings in other potato cultivars (Kruger et al., 1988). The presented results strongly suggest that the high molecular weight form (PFK1) represents cytosolic PFK and the low molecular weight form (PFK2) the plasid enzyme. In the first place, extracts of chloroplasts that are strongly related to amyloplasts (Isherwood, 1976; Ngernprasirtsiri et aI., 1988) contain only PFK2 (Fig. 5) and, secondly, the sensitivity of PFKl and PFK2 from leaves and tubers for phosphate and sulphate ions (Table 3) agrees with that of cytosolic and plastid PFK, respectively (Garland and Dennis, 1980; Balogh et al., 1984; Hausler et al., 1987 and 1989). Mature tubers of Bintje kept for 2 weeks at 17°C after harvest contain low levels of sucrose and reducing sugars. Transfer of these tubers to 2 °C results in an increase in sugar content and at the same time a decrease in the maximum activity of PFK1, while that of PFK2 remains fairly constant (Figs. 1 and 3). In addition, the activities of both PFK forms may be reduced due to the observed cold sensitivity (Fig. 4). A disproportional reduction in tuber PFK activity compared with other hexose phosphate metabolizing enzymes due to cold sensitivity of PFK has been reported by Pollock and ap Rees (1975) and by Hammond et aI. (1990). Thus the activity of the cytosolic PFKl in cold stored tubers seems to be reduced both by coarse control and by cold sensitivity, while that of the plastid PFK2 is only reduced by cold sensitivity. In addition, other factors such as effector concentrations (Dixon and ap Rees, 1980 b) and differences in affinity of PFK for its substrates and regulators may contribute to the response of PFK to changes in temperature (Hammond et al., 1990). Reduction of the activity of PFK in the cytosol diverts hexose phosphates from respiration to sucrose synthesis and could thus lead to accumulation of reducing sugars. When Bintje tubers are stored at 8°C, the amounts of sugars are fairly constant while the maximum activity of the PFK1 decreases, albeit much more gradually than during storage at 2°C. Consequently, there seems to be no correlation between sugar content and maximum activity of PFKl in tubers stored at 8°C, in contrast to tubers stored at 2 dc. Although the maximum activity, assayed at 25°C, decreases both during storage at 8°C and at 2 °C (Fig. 3), the actual activity of PFKl in tubers stored at 8 °C is apparently still sufficient to prevent sugar accumulation. Possibly, the differential reduction in activity of PFKl due to cold sensitivity is less at 8°C, due to protection by cytoplasmic solutes (Dixon et aI., 1981), although the breakpoint in the Arrhenius plots of PFKl is about 14°C. In addition, Ohad et aI. (1971) reported an increase in starch degradation in tubers stored at 2 dc. It seems reasonable that an increase in hexose phosphates may require a relatively higher PFK activity at 2 °C as compared to 8 dc. The changes in sugar content and activity of PFKl observed after storage of Bintje tubers at 2 °C are reversed when the tubers are transferred to a higher temperature. During this reconditioning a high proportion (80%) of the reducing sugar content is converted into starch and the remaining

and W. J.

STIEKEMA

20% is lost in respiration (van Es and Hartmans, 1987). The restoration of the maximum activity of PFKl may contribute to the latter process by facilitating glycolysis. In the case of KW74-2618 the results are generally similar to those of Bintje, except that the level of the maximum activity of PFKl in tubers of KW74-2618 is remarkably higher throughout the whole storage period and sugar accumulation is less extensive. This finding is consistent with the hypothesis that a higher PFKl activity confers a greater capacity to process hexose phosphates. Although PFKl and PFK2 from Bintje as well as from KW74-2618 showed cold-sensitivity in Arrhenius plots (Fig. 4), similarly as has been observed for PFK's from other potato cultivars (Pollock and ap Rees, 1975; Hammond et aI., 1990) these enzymes were not extraordinarily inactivated during incubation at 4°C as compared to 20°C. However, this discrepancy may have been caused by rapid reactivation of cold-inactivated PFK during the assay at 25°C (Angelopoulos and Gavalas, 1988). Dixon et ai. (1981) also assumed reassembly of dissociated PFK after return to a higher temperature. Yet it is still uncertain whether cold-inactivation indeed takes place in vivo, because it is known that cold-inactivation is dependent on an interplay of temperature with other factors such as pH and enzyme-protein concentration (Bock and Frieden, 1974; Angelopoulos and Gavalas, 1988). On the other hand, two lines of evidence support the view that cold-inactivation of PFK indeed plays a role in sweetening of cold-stored potatoes. First, the observation that some potato cultivars which do not show cold-induced sweetening have cold-stable PFK forms, while cultivars that sweeten have cold-sensitive PFK's (Hammond et aI., 1990) and secondly, the fact that the proportion of hexose-6-phosphates that enter respiratory pathways is reduced in the cold as established by the use of labelled substrates (Pollock and ap Rees, 1975; Dixon and ap Rees, 1980). At present it seems unclear whether the observed decrease in maximum activity of PFKl in tubers stored at 2 °C can be attributed to cold-inactivation alone, as the maximum activity of PFK2 does not decline, although this enzyme form is also cold-sensitive (Fig. 4). As PFKl activity also decreased in tubers stored at 8°C and 17°C it seems likely that this enzyme activity is additionally affected by other factors than cold. Possibly, cold-storage and sprouting, which has been observed in both genotypes at 8 and 17°C, both induce certain similar changes, e.g. in membranes, pH or effector concentrations, which in turn cause the decrease in maximum activity of PFKl. In that case, the effect of low temperature on maximum PFKl activity is indirect. In this context one can think of the concentration of A TP, which may regulate the activity of cytosolic carrot PFK by metabolite-dependent aggregation-disaggregation (Wong et aI., 1987) and which rises sharply after transfer of potato tubers to cold (Amir et aI., 1977). Preliminary experiments revealed that the pH of juice from tubers decreases from about 6.2 to 5.8 within 1 week of storage at 2 °C both in Bintje and KW74-2618; at 8°C the decline in pH takes place less rapidly and to a lesser extent. Small changes in pH may cause dissociation and concomitant inactivation of PFK in vivo (Carpenter and Hand, 1986). Whatever the cause of the decrease in maximum activity of PFK1, the presented results support the view that reduction

PFK in cold stored potato tubers

of PFK activity plays a role in sugar accumulation in cold stored tubers. On the other hand, cold-sensitivity of other glycolytic enzymes like glyceraldehyde phosphate dehydrogenase and pyruvate kinase may also contribute to reduction of the rate of glycolysis (Pollock and ap Rees, 1975). Acknowledgements The authors thank L. Colon for kindly providing the potato genotype KW74-2618.

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