Fructan synthesizing and degrading activities in chicory roots (Cichorium intybus L.) during field-growth, storage and forcing

j. Pl4nt Physiol. VOL 149. pp. 43-50 (1996)

Fructan Synthesizing and Degrading Activities in Chicory Roots (Cichorium intybus L.) during Field-growth, Storage and Forcing WIM VAN DEN ENDE

and ANmtE VAN WRE

Laboratory for Developmental Biology, Botany Institute, Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium Received October 8, 1995 . Accepted December 15, 1995

Summary

An investigation of SST (E.C. 2.4.1.99) and FFT (E.C. 2.4.1.100) activities in chicory roots (Cichorium intybus L. var flliosum cv. Flash) showed that both enzymes also had a small but genuine ~-fructosidase activity. The activity of fructan exohydrolase towards small and large fructans was comparable provided that they are present in the same molar concentration. During field growth the activity of SST decreased continuously to essentially disappear in October. FFT activities on the contrary remained high and even increased slightly. Fructan exohydrolase activity was detected throughout field-growth but in crude extracts a part of it is likely to be due to the ~-fructosidase activity of FFT. When this was taken into account the genuine fructan exohydrolase activity increased rapidly after mid-October. During cold storage a further rapid increase was detected. Forcing of the roots resulted in a considerable decrease of inulinase activity although large amounts of fructans are mobilized during that period.

Key words: Chicory, Cichorium intybus L., Asteraceae, inulin, Jructan, Jructan exohydrolase, inulinase, SST, FFT, ~-.fructosidase. Abbreviations: DP = degree of polymerization; FFT = fructan: fructan fructosyl transferase; MES = 2-(N-Morpholino)ethanesulfonic acid; SST = sucrose: sucrose fructosyl transferase. der Meer et al., 1994). The introduction of fructans in tobacco mediated enhanced resistance to drought stress Fructans are widely distributed as a carbohydrate reserve (Pilon-Smits et al, 1995). Three types of fructans can be discerned based on the among at least 10 families of higher plants, including the numerically and economically important Asteraceae and Po- binding of fructose units to one of the three primary aceae (Hendry, 1993). There is a great potential for the use of hydroxyl groups of sucrose. The ~-(2-1) linked fructans (inufructans as a raw material in a number of interesting food and lin) are common in the fleshy roots of chicory (Cichorium non-food applications (Fuchs, 1991; Hidaka, 1991). So far, a intybus L.), in the tubers of Jerusalem artichoke (Helianthus physiological role other than storage carbohydrate remains tuberosus) and several other Asteraceae (Meier and Reid, somewhat obscure. Fructans may play a role in tolerance to 1982). In their work on the latter species, Edelman and Jefcold stress (Pontis, 1989; Pollock, 1986) or in drought resist- ford (1968) suggested that inulin synthesis would be cataance (Hendry, 1993). An example of osmotic adaptation via lyzed by the concerted action of two fructosyl transferases. the use of fructans is the rapid conversion of fructans into SST would produce l-kestose and glucose from sucrose (G-F low-DP products as a mechanism to sustain petal expansion + G-F ~ G-F-F + G). FFT would be responsible for further in the daylily (Bieleski, 1993). Recently, both tobacco and chain elongation (G-Fn + G-F m <=> G-F(n_1) + G-F(m+l») (n>l, tomato plants were genetically transformed with bacterial m>O). Later, both enzymes and all fructans where shown to enzymes to synthesize fructans (Ebskamp et al., 1994; Van be vacuolar (Wiemken et al., 1986; Darwen and John, 1989). Introduction

© 1996 by Gustav Fischer Verlag, StUttgart

44

WIM VAN DEN ENDE and ANDRE VAN WRE

Fructan catabolism is believed to be regulated by the combined action of FFT together with fructan exohydrolase (Edelman and Jefford, 1968; Wiemken et al., 1986). We became interested in the metabolism of inulin in Cichorium intybus 1. as part of a project to elucidate the physiology of widoof production. The production of witloof or Belgian endives encompasses two or three different phases. In a first step, biennial plants of Cichorium intybus 1. plants are grown in the field as rosette plants that develop a tap root, the weight of which exceeds the weight of the foliage at the end of the growing season. Before winter, the plants are uprooted, the leaves are cut off and the roots are stored for a short (or prolonged) period at 1 "C and a relative humidity of 90-98 %. Thereafter, the roots are allowed to develop new etiolated leaves in the dark at temperatures around 16"C. This can be done either in soil by covering the plants with top soil or, more recently, in hydroponic culture in dark rooms. Since, in both cases, the chicon develops in complete darkness, the materials needed for endive production are mobilized from the roots. We were able to purify fructan exohydrolase (Claessens et al., 1990), SST (Van den Ende and Van Laere, 1993), FFT (Van den Ende et al., unpublished results) and neutral invertase (Van den Ende and Van Laere, 1995) from chicory roots. The SSTIFFT model for synthesis of fructan was subjected to severe criticism (Cairns, 1993). Recently, however, we provided evidence to validate the «two enzymes, one compartment» model, at least in the Asteraceae. Indeed, a mixture of purified chicory root SST and FFT was able to synthesize authentic chicory root fructan in vitro starting from physiologically relevant sucrose concentrations (Van den Ende et al., unpublished results). The dynamics of inulin synthesis and breakdown during chicory root development, storage and forcing have been described (Rutherford and Weston, 1968; Bhatia et al., 1974; Rutherford and Phillips, 1975; Limami and Fiala, 1991; Van den Ende et al., unpublished results). This paper reports on the activities of SST and FFT during the development of chicory plants and the fructan exohydrolase activities in fieldgrown chicory roots during autumn and during two different storage and forcing periods. Also the substrate specificity of purified chicory root inulinase is reported.

Material and Methods

Plant material

Cichorium intybus L. (var. flliosum cv. Flash) was sown in a local field with sandy loam soil on June 1, 1994. On a weekly basis, nine plants were uprooted to investigate fructan synthesizing Ouly 26thNovember 3rd) and/or fructan degrading (September 13th-December 6th) activities. On two different dates (October 4th and October 25th) a number of plants were uprooted, the leaves were cut off about 5 cm above the root collar and the roots were stored at +1 ·C for 3 weeks. After this cold sto!age, roots were forced for 3 weeks at 16·C in a hydroponic system containing tap water supplemented to contain 5.52 mmol/L Ca2 +, 2.0 mmol/L MI?+, 13.65 mmol/L K+, 14.5mmol/L N03 -, 2.15mmol/L pol-, and 2.7mmol/L sol(pH 6.8). During storage and forcing, three roots were analyzed at regular intervals (at least once a week).

Extraction Roots were washed with cold tap water, peeled and cut radially to obtain a 10 g (field-grown chicory roots) or a 30 g (stored or forced roots) sample over the whole length of the root (limited to 18 cm from the top). Tissue from three roots (3 X 10 g, field-grown chicory roots) or from one root (30 g, stored or forced roots) was homogenized for 1 min with a Waring blender in 30 mL ice-cold 50 mmol/L Na-acetate buffer, pH 5, containing 0.02 % (w/v) Na-azide, 1Ommol/L NaHS0 3, 1 mmol/L phenylmethyl-sulfonylfluoride, 1 mmol/L mercaptoethanol and 0.1 % Polyclar (Serva, Heidelberg, Germany), Three independent homogenates were prepared. They were centrifuged for 3 min at 3,000 gn. The supernatant was centrifuged again for 2 min at 10,000 gn. This supernatant was used as the source of SST, FFT and fructan exohydrolase activities (see below).

Assay ofSST and FFT Two aliquots of 2 mL from the 10,000 gn supernatant were mixed with 4.65 mL saturated ammonium sulfate. After 30 min at 0 ·C the precipitate was collected by centrifugation at 40,000 gn for 15 min. The precipitate was suspended in 80 % ammonium sulfate and centrifuged again under the same conditions to further reduce sugar and fructan contents. The precipitates were dissolved in 0.5 mL 50 mmol/L Na-acetate buffer, pH 5, containing 0.02 % (w/v) Naazide (acidic protein extract) or in 0.5 mL 50 mmol/L MES-NaOH buffer, pH 6.2, containing 0.02 % (w/v) Na-azide (neutral protein extract) and centrifuged for 2 min at 10,000 gn. SST activity was measured by incubation of the supernatant of the acidic protein extract for 1 h at 30 ·C in 50 mmol/L Na-acetate buffer, pH 5, containing 100 or 20 mmollL sucrose and 0.02 % (w/v) Na-azide. The reaction was stopped by heating at 95 ·C for 5 min. Samples were diluted threefold with 0.03 % (w/v) Na-azide. From these, 25 j.LL was automatically injected onto a Dionex column (see below). Zerotime reaction mixtures (blanks) only contained sucrose and traces of glucose and fructose. Glucose, fructose and l-kestose were the only reaction products formed. SST activity is expressed in nmoles of l-kestosemin-IgFW-l. FFT activity was measured by incubation of the supernatant of the neutral protein extract for 15 min at O·C in 50 mmol/L MESNaOH buffer, pH 6.2, containing 10 mmol/L l-kestose and 0.020/0 (w/v) Na-azide. The reaction was stopped by heating at 95 ·C for 5 min. Samples were diluted threefold with 0.03 % (w/v) Na-azide. From these, 25j.LL was automatically injected onto a Dionex column (see below). Zero-time reaction mixtures (blanks) only contained l-kestose and traces of sucrose, glucose and fructose. FFT activity is expressed in nmoles of 1, I-nystosemin-1gFW-1.

Assay offructan exohydrolase In order to remove small endogenous carbohydrates, 1 mL of the 10,000 gn supernatant was loaded onto a Fast Desalting column (Pharmacia HR 10/10) equilibrated with 50 mmollL Na-acetate buffer, pH 5, containing 0.02 % (w/v) Na-azide. The flow rate was 1 mL min -I, Since proteins eluted first from the column as a single peak, a fraction of exactly 2.5 min was taken (starting from the first increase of the OD 28o) The exact volume of this fraction (about 2.5 mL) was used to calculate the correct dilution. Fructan exohydrolase activity was measured by incubation of this fraction together with 1 % of commercial chicory root inulin (Sigma) in 50 mmollL Na-acetate buffer, pH 5, containing 0.02 % (w/v) Naazide at 30 ·C for 2 h. The reaction was stopped by heating at 95 ·C for 5 min. From these, 25j.LL was automatically injected onto a Dionex column (see below). Fructan exohydrolase activity is expressed in nmoles of fructose min-1gFW-1.

45

Fructan synthesizing and degrading activities in chicory roots

Carbohydrate analysis Carbohydrates were analyzed by HPLC (Dionex, Sunnyvale CA, USA) on a CarboPac PAl anion-exchange column (Shiomi et al., 1991) and quantitated by a pulsed amperometric detector equipped with a gold electrode (potentials: E I: +0.05 V; E2: +0.6 V; E3: -0.8Y). The flow rate was ImLmin-l . The SST samples were run isocratically with 90 mmollL NaOH and 50 mmollL Na-acetate. The FFT samples were analyzed using 90 mmollL NaOH and 75 mmollL Na-acetate. The same conditions as for the SST samples were used for the fructan exohydrolase samples (10 min) except that afterwards the inulin was washed from the column using 1 M NaOH for 10 min. In the latter assay the column was equilibrated with 90 mmollL NaOH and 50 mmollL Na-acetate for 20 min after every run. Quantification was performed on the peak areas with the external standards method for glucose, fructose, sucrose, l-kestose and 1,I-nystose. Only the peaks exceeding the baseline noise by a factor of 20 were taken into account.

Preparation ofsubstrates l-kestose and 1, I-nystose were prepared from Neosugar P by preparative reversed phase HPLC (Nucleosil 7 CIS, 250 X 12.7 mm) with water as solvent and a flow rate of 2 mL min -I. Manually collected fractions were pooled and lyophilized. Cichorium intybus L. roots were used as a source for the preparation of an «oligofructan mixture» mainly consisting of DP6-1O. Therefore, 100 g of root tissue were homogenized with a Waring blender in 50 mL acetone. The homogenate was squeezed through cheese cloth. The filtrate was centrifuged for 30 min at 20,000/{n. The supernatant was dried in a Speed Vac and redissolved in water. Carbohydrates with a DP<6 were removed by preparative reversed phase HPLC (Nucleosil 7 CIS' 250 X 12.7 mm) with 1.5 % methanol as solvent and a flow rate of 2 mL min -I. Then a broad peak eluted from the column containing a mixture ofDP6 (24%), DP7 (19%), DP8 (19 %), DP9 (18 %), DPI0 (12 %), DPll (3 %), DP12 (0.5 %), DP13 (0.4 %) and some other compounds (4.1 %) probably representing inulonoses. A mean DP of 8 was calculated. This fraction was dried in a Speed Vac concentrator and redissolved in water. It is referred to as «oligofructan mixture» throughout this paper. A mean DP of 33 was used for commercial chicory root inulin (Sigma, DP~lO) in our calculations (Bonnett et al., 1994).

Results

Some preliminary experiments on SST and inulinase properties We followed the pattern of fructan during growth in the field, storage and forcing during several years (Van den Ende et al., unpublished results). In order to understand inulin metabolism, the observed in vivo pattern has to be correlated with in vitro measurements of relevant enzyme activities. Although we were able to obtain some rough data of fructan exohydrolase activities during the 1990 growing season (Claessens, Ph. D. thesis, 1993) and also from SST during the 1993 growing season, we found it useful to measure both fructan synthesizing (SST and FFT) and degrading (fructan exohydrolase) enzyme(s) in the same year. Therefore, some important preliminary questions needed to be taken into account. What are the optimal reaction conditions needed (pH, temperature, choice of substrate) for the three enzymes investigated? Is it possible to choose conditions so that interference from other enzyme activities is minimized? Recently, FFT has been purified and fully characterized (Van den Ende et al., unpublished results). l-kestose was found to be the very best fructosyl donor for the enzyme. The enzyme also exhibited ~-fructosidase activity, especially at higher temperatures and lower substrate concentrations. The synthesis of fructan from l-kestose decreased at higher temperatures (5-50°C). Therefore, FFT activity measurements were performed at O'C with l-kestose as the only substrate. The reaction time was chosen rather short (15 min) to prevent synthesis of fructans greater than DP4. Since fructose was also formed by SST (Van den Ende and Van Laere, 1993; Van den Ende and Van Laere, unpublished results) we also investigated the ~-fructosidase (or invertase) activity of purified chicory root SST as a function of the incubation temperature. The temperature optimum of purified chicory root SST is presented in Fig. 1, showing maximal production of

100

Substrate specificity ofpurified jructan exohydrolase Fructan exohydrolase was purified as described by Claessens et al. (1990) using field-grown chicory roots in November, 1994. The enzyme was stabilized by the addition of 30 % ethylene glycol. Aliquots of the enzyme were incubated together with several concentrations of sucrose, l-kestose, 1,I-nystose, the oligofructan mixture and commercial chicory root inulin in 50 mmollL Na-acetate buffer, pH 5, containing 0.02 % (w/v) Na-azide for 2 hat 30°C.

Temperature optimum ofSST SST was purified according to the purification scheme of Van den Ende and Van Laere (1993) except that the last purification step (Mono Q pH 8) was replaced by a run with Mono S pH 4 (Pharmacia HR 5/5 equilibrated with 20 mmollL Na-acetate buffer, pH 4, and 10 % (v/v) ethylene glycol; fractions of 1 mL were collected using a linear gradient from 0 to 0.3 M NaCl in 30 min and a flow rate of 1 mLmin-I). Aliquots of the enzyme were incubated together with 100 mmollL sucrose in 50 mmollL Na-acetate buffer, pH 5, containing 0.02 % (w/v) Na-azide for 20 min at 0, 5, 10, 15, 20, 25, 30, 40 and 50·C. Both enzyme and substrate were first allowed to come to the desired temperature for 15 min.

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46

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l-kestose from sucrose at 30·C. Although fructose production (expressed as a percentage of l-kestose) increases at higher temperatures, ~-fructosidase (or invertase) activities remain rather low (Fig. 1). Since activities at 0 ·C were only 20 % of ~he activities at 30 ·C, the benefit of the loss of ~-fructosidase activity at O·C was considered less important than the major loss of SST activity at O·C. Therefore, SST was assayed at 30·C. Chicory root fructan exohydrolase was purified and partially characterized (Claessens et al., 1990). However, no data concerning substrate specificity were available. Does the enzyme have a higher or lower affinity towards low DP fructan? Does the enzyme also have invertase activity? To resolve these questions the enzyme was purified and incubated with varying concentrations of sucrose, l-kestose and 1,I-nystose (Fig. 2A). Like FFT, fructan exohydrolase has no activity against sucrose as substrate. Neither fructan exohydrolase, nor FFT are therefore expected to interfere in the SST assay. Fructose production from different concentrations of the oligofructan mixture (DP6-DPI0) and from commercial chicory root inulin (DP~lO) are shown in Fig. 2B. Although results from

100

Fig. 3: The actlVlty of A, SST (.. , 100 mmollL; ~, 20 mmollL sucrose as substrate) and B, FFT in extracts from chicory roots during field-growth Guly 26thNovember 3rd, 1994). Bars represent standard errors for n=3.

Fig. 2 suggest some differences in kinetic parameters (Km, Vmax> for the different fructan investigated, a comparison of activities at a concentration of about 3 mmollL revealed no major differences between the fructan. Therefore, commercially available chicory root inulin was chosen as a substrate since the preparation of l-kestose, 1, I-nystose or oligofructan is expensive and time-consuming. Moreover, it was verified that the enzyme showed no affinity differences between selfprepared chicory root inulin and commercial chicory root inulin.

Activities ofSST and FFT during the growing season l-kestose production obtained from incubation of the acid protein extract together with 20 or 100 mmol/L sucrose throughout the growing season Guly 26th-November 3rd) is presented in Fig. 3 A. SST activity clearly decreases gradually in function of time. The ~-fructosidase activity (fructose production as a percentage of the l-kestose production) was nearly constant throughout the growing season. Means and standard errors of the latter activity for 20 and 100 mmol/L

47

Fructan synthesizing and degrading activities in chicory roots 30

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sucrose were 38.72 ± 1.69% and 9.18 ± 1.26%, respectively. The I-kestose production from 20 mmol/L sucrose as a percentage of the I-kestose production from 100 mmollL sucrose also was very constant throughout the growing season. Mean and standard error were 26.78 ± 1.08. Fig. 3 B shows the I, I-nystose production from the neutral protein extract and I-kestose over the same period. No important activity changes were observed. However, there was a tendency to increase, especially at the end of the growing season.

Activities offructan exohydrolose during growth, storage and forcing Fructose production from protein extracts and I % commercial chicory root inulin during the autumn of the 1994 growing season is presented in Fig. 4. Surprisingly, we already found fructose production from inulin on September 13th.

Since we could never purify fructan exohydrolase (Claessens et al., 1990) before October, we became suspicious about the observed fructose production and whether if FFT could be responsible for this observation. Indeed, protein extracts also contained considerable FFT activity in the autumn (Fig. 3 B) and purified chicory root FFT exhibited considerable ~-fruc­ tosidase activity towards lower concentrations of I-kestose at 30·C (Van den Ende et al., unpublished results). Therefore, the ~-fructosidase activity of FFT was assayed against the rather low inulin concentration (1 % or about 1.86 mmollL) used in the fructan exohydrolase assay. This was done by measuring the FFT activity (1,I-nystose production from l-kestose) of a crude extract. Afterwards, a similar reaction mixture was prepared but boiled immediately. After cooling, purified FFT was added to obtain a similar FFT activity as in the crude extract and the fructose production from inulin by FFT was 8.79 % of the I,I-nystose production from 10 mmollL I-kestose on September 13th. If one assumes that this factor remains constant throughout the growing season, one can subtract the fructose production by FFT from the total hydrolase result to obtain the real fructan exohydrolase activity (Fig. 4, open symbols). Consequently, an eightfold increase was obtained for the activity of fructan exohydrolase alone: the increase only became really significant after October 15th. Fructose production from inulin sharply increased during the first days of the first storage period. Afterwards, activities stabilized (Fig. 5A). Upon forcing, a very important decrease was found (Fig. 5 A). At the beginning of the second storage period inulinase activity was already high but increased even further over 2 weeks of cold storage (Fig. 5 B). Like during the first forcing period, activities decreased again even below the activity level obtained from field-grown roots. Since we did not measure FFT activities during storage and forcing, the observed fructose production possibly is the result from both FFT and fructan exohydrolase activities.

Discussion

So far, work on seasonal changes in the biochemistry of fructan storing organs has been largely focused on the exami-

40

t:6 Fig. 5: Fructose production from 1 % com-

mercial chicory root inulin by chicory root extracts during cold storage (up to the arrow) and forcing (after the arrow) . A, first storage and forcing period (October 4thNovember 15th); B, second storage and forcing period (October 25th-December 6th). Bars represent standard errors for n=3.

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48

WIM VAN DEN ENDE

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nation of changes in the stored carbohydrates (see also Introduction). Although SST, FFT and fructan exohydrolase were properly described and characterized from a number of plant species, detailed information about their activities during development are rather scarce in the literarure. SST activity gradually decreased throughout the growing season (Fig. 3 A). At the time SST activity is high, fructans are rapidly synthesized (Van den Ende et al., unpublished results). SST activities disappeared almost completely in October (Fig. 3 A) and also during storage and forcing of the roots (Van den Ende et al., unpublished results). As already discussed by Edelman and Jeff"ord (1968) in their work on Heiianthus tuberosus, SST may be the controlling factor for the rate of growth of the chicory root since its activity disappears rapidly when the roots stop growing. Moreover, there is a very good correlation with the concentrations of free glucose (one of the SST products) in the roots (Van den Ende et al., unpublished results). Upon termination of root growth the decline in free glucose could characterize the transition of the root from a sink to a source. Limami and Fiala (1991) questioned SST as a controlling factor for the rate of growth since cleared homogenates from both growing (fructan accumulatinw and mature chicory roots produced 4C)-1-kestose from (U 4C)-sucrose in vitro. Since their cleared crude homogenates still contained all endogenous fructans, the observed production of 4C)-1-kestose from marure root extracts is likely to be caused by the FFT catalyzed fructosyl transfer from inulin to (U I4C)-sucrose. Indeed, sucrose has been found to be the best acceptor for FFT (Koops and Jonker, 1994; Van den Ende et al., unpublished results) and its activity showed a tendency to increase during maturation of chicory (Fig. 3 B). Also Gupta et al. (1985) found a decrease in fructosyltransferase activity between l.5 and 4.5 months afrer sowing. Unlike Limami and Fiala, they incubated dialyzed ammonium sulfate precipitates (no endogenous fructans) together with (U I4 C)-sucrose. The observed (14C)-1-kestose production continuously decreased between l.5 and 4.5 months afrer sowing. Both SST and FFT activities were also measured by Shiomi (1992) in his work on Asparagus roots. Over the period investigated SST activity first increased to a maximum and then decreased continuously. As is the case in chicory roots (Fig. 3 B), FFT activity was higher than SST activity and nearly constant over the period investigated. These observations confirm the original model from Edelman and Jeff"ord (1968) in which FFT fulfills a key role during both fructan synthesis and breakdown. We found that the (3-fructosidase activity of SST (expressed as a percentage of the 1-kestose production) was constant throughout the growing season (both for 20 and 100 mmollL sucrose). Moreover, the observed (3-fructosidase activity was not much higher than the one obtained from purified SST (Van den Ende and Van Laere, unpublished results). Therefore, we are confident that the SST activities measured from the ammonium sulfate precipitates are due to the same SST that was purified afrer Mono S kation exchange chromatography. We are convinced that acid invertase activity, if present, could only be very small compared with SST activity. SST activity ratios with 20 and 100 mmollL sucrose also were constant throughout the growing season, suggesting that the Km of the enzyme towards sucrose did not change.

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e

So, probably there is no modification of SST at the end of the growing season, as first suggested to explain the loss of SST activity of the invertase/SST preparation (Van den Ende and Van Laere, 1993). Indeed, we could recently purify a small amount of an authentic acid invertase (producing only glucose and fructose from various sucrose concentrations) from chicory roots, indicating that the original invertase/SST preparation could have been a mixture of both SST and an acid invertase. Nevertheless, our recently purified SST preparations still catalyzed some fructose production from lower sucrose concentrations. SST was found to be electrophoretically pure on NATIVE-PAGE. On SOS-PAGE both 49 and 20 kDa bands were found, suggesting that the enzyme is a heterodimer (Van den Ende et al., unpublished results). Both the 49 and 20 kDa fragments gave only one signal during N-terminal sequencing, proving the purity of our SST preparation. Therefore we are now convinced that the observed remaining (3-fructosidase activity is a real property of SST. In their work on the seasonal changes in the invertase and hydrolase activities of Jerusalem Artichoke tubers, Rutherford and Flood (1971) reported a very large increase in invertase activity at a time when inulin synthesis was most active. As the rate of inulin synthesis decreased so too did the invertase activity. When the tubers became dormant, very little activity of invertase could be detected. They measured invertase activities by incubating protein extracts (free from endogenous substrates) together with 20 mmol/L sucrose and quantified the reducing sugar formation. Since their results are in close agreement with our and other SST results, possibly they were (at least pardy) measuring SST activity. As for SST, purified FFT also exhibits (3-fructosidase activity at low substrate concentrations. In doing so it can interfere with in vitro measurements of fructan exohydrolase in crude extracts (Fig. 4). Whether the observed ~fructosidase activity of FFT also is important in vivo is unclear. Whatever the outcome of this question may be, it is clear that the hydrolase activity in the roots increases afrer October 15th (Fig. 4). This correlates very well with the observed changes in carbohydrate concentrations found around that time during several previous growing seasons: a five-fold increase in fructose concentration and a breakdown of high OP fructans (Van den Ende et al., unpublished results). However, the observed shift from high OP fructans to lower OP fructans could also be (partly) due to the action of FFT using low molecular weight carbohydrates as acceptors. The hydrolase activity in Heiianthus tuberosus increased throughout the period of ruber formation and was maximal during the initial stages of dormancy (Rutherford and Flood, 1971). Cold storage results in a rapid depolymerization of large fructans with a simultaneous increase in smaller fructans, sucrose and fructose (Rutherford and Jackson, 1965; Rutherford and Weston, 1968; Van den Ende et al., unpublished results). The increase of hydrolase activity during the two storage periods investigated correlates very well with these carbohydrate changes. Also, Rutherford and Phillips (1971) found an increase in hydrolase activities during storage. Forcing of chicory roots and concomitant chicon development was accompanied by an even faster breakdown of fructans and an accumulation of fructose (Rutherford and Phil-

Fructan synthesizing and degrading activities in chicory roots lips, 1975; Van den Ende et al., unpublished results). However, a considerable decrease of the hydrolase activity was observed (Fig. 5 A, B). This decrease was not reported by Phillips and Rutherford (1976) but, in contrast with our work, they used a DP5-DP12 oligofructan mixture as a substrate. On first sight our results seem contradictory with the observed fructose upsurge during forcing. A reasonable but speculative explanation to overcome this discrepancy is the replacement of the field-induced fructan exohydrolase by another one with preferential activity towards small fructans. Further investigation on the hydrolases in forced (or sprouting) chicory roots is needed to resolve these problems. Since average values of 20 j.lmol sucrose (g FW)-l and 10 j.lmol 1-kestose (gFW)-1 were measured during the period of active fructan synthesis Ouly 12th-September 27th) in the 1993 growing season (Van den Ende et al., unpublished results), both SST and FFT were assayed at roughly in vivo concentrations. It is obvious that the SST/FFT activity ratio changes enormously throughout the growing season (from 0.636 on July 26th to 0.016 on November 3rd). We could recently demonstrate the de novo synthesis of authentic chicory root fructans from sucrose in vitro by a combination of purified SST and FFT, strongly suggesting the in vivo relevance of these enzymes (Van den Ende and Van Laere, unpublished results). Since the SST/FFT ratio decreases, fructan synthesis is bound to reach a plateau. The disappearance of SST probably enhances the competitive inhibition by sucrose as an acceptor for FFT; FFT might then start to depolymerize larger fructans to smaller ones by fructosyl transfer to sucrose and therefore fulfill a role in fructan catabolism too. Acknowledgements

The authors would like to thank E. Nackaerts for valuable technical assistance. W. Van den Ende is grateful to the National Fund for Scientific Research (NFSR Belgium) for a grant as research assistant.

References BHATIA, I. S., S. K MANN, and R. SINGH: Biochemical changes in the water-soluble carbohydrates during the development of chicory (Cichorium intybus L.) roots. J. Sci. Agric. 25, 535 - 539 (1974). BIELESKI, R. L.: Fructan hydrolysis drives petal expansion in the ephemeral daylily flower. Plant Physiol. 103,213-2190993). BONNETI, G. D., I. M. SIMS, J. A. ST. JOHN, and R. J. SIMPSON: Purification and characterization of fructans with ~-2, 1- and ~-2,6-glycosidic linkages suitable for enzyme studies. New Phytol. 127, 261-269 (1994). CAIRNS, A. J.: Evidence for the de novo synthesis of fructan by enzymes from higher plants: a reappraisal of the SSTIFFT model. New Phytol. 123, 15-24 (1993). CLAESSENS, G., A VAN WRE, and M. DE PROFT: Purification and properties of an inulinase from chicory roots. J. Plant Physiol. 136, 35-39 (1990). DAR-WEN, C. W E. and P. JOHN: Localisation of the enzymes of fructan metabolism in vacuoles isolated by a mechanical method from tubers of Jerusalem artichoke (Helianthus tuberosus L.). Plant Physiol. 89, 658-663 (1989).

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EBSKAMP, M. J. M., I. M. VAN DER MEER, B. A SPRONK, P. J. WEISBEEK, and S. C. M. SMEEKENS: Accumulation of fructose polymers in transgenic tobacco. Bio/Technology 12, 272-275 (1994). EDELMAN, J. and T. G. J EFFORD: The mechanism of fructan metabolism in higher plants as exemplified in Helianthus tuberosus. New Phytol. 67, 517-531 (1968). FUCHS, A: Current and potential food and non-food applications of fructans. Biochem. Soc. Trans. 19,555-560 (1991). GUPTA, A. K, MAMTA, and I. S. BHATIA: Glucofructosan metabolism in Cichorium intybus L. Phytochemistry 24, 1423-1427 (1985). HENDRY, G.: Evolutionary origins and natural functions of fructans. A climatological, biogeographic and mechanistic appraisal. New Phytol. 123,3-14 (1993). HIDAKA, H., M. HIRAYAMA, and K YAMADA: Fructooligosaccharides. Enzymatic preparation and biofunctions. J. Carbohydr. Chern. 10,509-522 (1991). Koops, J. and H. H. JONKER: Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus
SO

WIM VAN DEN ENDE and ANDRE VAN WRE

VAN DEN ENDE, W. and A. VAN WRE: Purification and properties of an invertase with sucrose: sucrose fructosyltransferase (SSn activity from the roots of Cichorium intybus L. New Phytol. 123,

31-37 (1993).

-

- Purification and properties of a neutral invertase from the roots of Cichorium intybus. Physiol. Plant. 93, 241-248 (1995).

VAN DERMEER, I. M., M.J. M. EBSKAMP, R. G. F. VISSER, P.J. WEISBEEK, and S. C. M. SMEEKENS: Fructan as a new carbohydrate sink in transgenic potato plants. The Plant Cell 6, 561-570 (1994). WIEMKEN, A., M. FREHNER, F. KELLER, and W. WAGNER: Fructan metabolism, enzymology and compartmentation. Curro Top. Plant Biochem. Physiol. 5, 17-37 (1986).