Effect of osmolytes on the fructan pattern in feeder roots produced during forcing of chicory (Cichorium intybus L.)

• JOU •• ALO'.

Plan. Pb,•••I••,

© 1998 by Gustav Fischer Verlag. Jena

Effect of Osmolytes on the Fructan Pattern in Feeder Roots Produced during Forcing of Chicory (Cichorium intybus L.) WIM VAN DEN ENDE, SAM MOORS, GERD VAN HOENACKER,

and ANDRE VAN WRE

Laboratory for Developmental Biology, Botany Institute, KULeuven, Kardinaal Mercierlaan 92, B-300l Heverlee, Belgium Received June 26, 1997· Accepted September 26, 1997

Summary

During forcing of chicory tap roots (Cichorium intybus 1. var. flliosum cv. Final) for witloof production in hydroponics a large number of thin feeder rootsis formed. An inulin pattern of fructans (up to DP20) accumulated in these roots. The roots were also subjected to osmotic stress by addition of 0.2 molll NaCI or a PEG 6000 solution with the same water potential. Both addition of NaCl or PEG resulted in an increase of internal browning in the heads of witloof chicory, the most important disorder considerably reducing the market value of the chicon. The 0.2 molll NaCl treatment resulted in an important reduction in lateral root and chicon growth; a slightly increased fructan concentration in these feeder roots was also observed. PEG treatment was equally inhibitory on chicon growth, but feeder root growth was nearly not affected. Much lower fructan concentrations were found in these roots but the fructose, glucose and sucrose concentrations were much higher, apparently increasing the osmotic contribution of the stored carbohydrates in the feeder roots. It was demonstrated that this PEG-mediated drought stress effect has nothing to do with hypoxia. The dynamics of carbohydrates during forcing are discussed based both on the determination of enzymatic activities (invertase, I-SST, I-FFT and I-FEH) and the composition of the feeder root phloem exudate.

Key words: Chicory, Cichorium intybus L., Asteraceae, I-FEH, I-FFT, I-SST, chicon, feeder roots, forcing, fructan, internal browning, inulin, invertase, osmotic stress, sucrose. Abbreviations: I-FEH

=fructan l-exohydrolase; I-FFT =fructan: fructan I-fructosyl transferase; I-SST

=sucrose: sucrose I-fructosyl transferase; DP =degree of polymerisation; PEG =polyethyleneglycol. Introduction

The production of the etiolated buds (chicons or Belgian endives) from the tap roots of witloof chicory (Cichorium intybus 1. var. flliosum) has a long tradition in Belgium and still is economically very important. Nowadays, the forcing process mainly occurs in controlled growth chambers (dark, relative humidity 95-98 %, air temperature 14-20°C) using a recirculating hydroponic solution. During the forcing process both an uptake of water (a chicon contains roughly 90 % water) and a mobilisation of reserves from the tap root are necessary. Since the harvested and stored roots are suberised at their surface, water uptake is largely dependent on the formation

J Plant PhysioL WlL 153. pp. 290-298 (1998)

of new lateral roots. As a reserve material, the tap root contains large amounts of fructans. Enough evidence is now provided to support the originall-SSTI I-FFT model (Edelman and Jefford, I968) for the synthesis of inulin-type fructans (Koops and Jonker, 1996; liischer et al., 1996; Van den Ende and Van Laere, 1996 a). During cold storage and forcing, these fructans are partly depolymerised (Van den Ende et al., 1996b) by I-FEH (Claessens et al., 1990; Van den Ende and Van Laere, 1996 b) and perhaps by I-FFT (Van den Ende et al., 1996 a, c) since a lot of transportable carbohydrate is needed for export to the developing chicon and the numerous feeder roots developing during the process. A possible role of fructans in osmoregulation has been discussed by Hendry (1993). A complete fructan depolymerisa-

Chicory Fructans: Osmotic Effects

tion has been implicated as the driving force for water uptake during petal expansion of Hemerocallis flowers (Bieleski, 1993). From literature, it is not straightforward whether a polymerization or depolymerization of fructans occurs as a result of drought stress. In tall fescue leaves, an induced water deficit resulted in a partial hydrolysis of fructans. A 70 % less direct contribution of low DP fructan to osmotic potential was observed while the contribution of sucrose and hexose increased 96 and 67 %, respectively (Spollen and Nelson, 1994). In contrast, however, under PEG mediated drought stress a strong increase in fructan concentration (fructan polymerization) was observed in transgenic tobacco producing bacterial fructans (Pilon-Smits et al., 1995). In this paper, we therefore investigated whether such an enhanced fructan polymerization reaction could also be observed in the lateral feeder roots subjected to drought stress by strongly manipulating the water potential of the hydroponic solution (by using NaCl or PEG). As a second point of our interest, in view of the economical importance, the effect of drought stress on the yield and quality of the chicons produced were also investigated.

Materials and Methods

Plant material and forcing conditiom Cichorium intybus L. (var. flliosum cv. Final) was grown on sandy loam soil in a local field sown on May 23,1995. On November 8th plants were uprooted, the leaves were cut off about 5 cm above the root collar and the roots were stored at -1 DC until July 10, 1996. Mter this cold storage, four times 40 roots of about the same size were chosen and subsequently forced in the dark for 23 days at 14 DC in a recirculating hydroponic system using four different solutions: A: water containing 122mglL Ca2+, 15.6mglL Mg2+, 17.8mglL Na+, 3mg/L K+, 60mglL and 42mglL Cl- (control); B: water as in A supplemented with 0.1 mollL NaCl ('I' = -0.477MPa); C: water as in A supplemented with 0.2 mollL NaCI ('I' = -0.954 MPa) , and D: water as in A supplemented with 0.035 mollL PEG-6000. The PEG concentration was calculated to be iso-osmotic with the 0.2 mollL NaCl treatment using the data of Money (1989). The electric conductivity (EC) of the hydroponic solutions was held at a constant level. It was verified that the pH of the solution was at a constant level (pH 7.2). From each condition, four roots were taken for analysis at least twice a week. To reduce complexity, only the data obtained after 6, 16 and 23 days of forcing were tabulated.

sol-

Extraction Roots and new feeder roots were washed with cold tap water. The developing chicon and feeder roots were removed and weighed. The length of the chicon was also recorded. From each root, tissue from feeder roots (1 g) was homogenised with mortar and pestle in 2 mL of ice-cold 50 mmollL Na-acetate buffer at pH 5.0 containing 10 mmollL NaHS0 3, 1 mmollL phenylmethylsulfonylfluoride, 5 mmol/L mercaptoethanol and 10 mmollL mannitol (as internal standard).

Carbohydrate analysis One g of this homogenate was diluted fivefold with water and put in a boiling water bath for 20 min. After cooling to room temperature the extract was centrifuged at 3000 Kn for 5 min. A 1 mL

291

sample of the supernatant was passed through a 1 mL bed volume of Dowex-50 H+ and a 1 mL bed volume of Dowex-l-acetate. The resins were rinsed four times with 1 mL distilled water. From this neutral fraction 50 ILL were analysed by HPLC (Waters, Milford MA, USA) on a Carbo Pac PAl (Dionex, Sunnyvale, Ca) anion-exchange column and quantitated by a pulsed amperometric detector equipped with a gold electrode (potentials: E 1: +0.05V; E2: +0.6V; E3: -0.8Y). The flow rate was ImLmin-l . The elution conditions were 90 mmollL NaOH with 30 mmol/L Na-acetate for 1 min followed by a linear gradient from 30 to 350 mmollL Na-acetate in 90 mmollL NaOH over a 45 min period. The column was regenerated with 1 mol/L NaOH for 10 min and equilibrated for 20 min after every run. Quantification was performed using mannitol as an internal standard. Conversion factors (relative to mannitol) of glucose, fructose, sucrose, l-kestose and 1,I-nystose were obtained by using the external-standard method. Since the higher oligofructans (DP >4) were not available, their response coefficients were estimated using the acta ofCharterton et al. (1993). Only the peaks exceeding the baseline noise by a factor of 10 were considered. After 21 days of forcing, individual feeder roots (obtained from 4 tap roots forced in the control hydroponic solution A) had an average length of 30 cm. Individual feeder roots were divided into proximal (O-lOcm), medial (10-20cm) and distal (>20cm) parts. In these three parts and also for the tap root both the carbohydrate concentrations (see higher) and enzymatic activities of invertase, I-SST, I-FFT and I-FEH (see below) were measured. We did not have enough root material for appropriate enzyme determinations for conditions B and C. For condition D it was impossible to obtain intact individual feeder roots due to the complex interweaving of these highly branched roots (from many different tap roots) under these conditions. At the end of the forcing period, the length of the centta! axis in the chicon and the occurrence of internal browning (core browning) were also recorded.

Enzymatic activities Tissue from 1 g of feeder roots was homogenized as described above. Part of the extract was centrifuged for 5 min at 10,000 Kn. Saturated ammonium sulfate was added to the supernatant to a final saturation of 80 %. After 30 min at 0 DC the precipitate was collected by centrifugation at 10,000 Kn for 10 min. The precipitate was suspended in 80 % ammonium sulfate and centrifuged again under the same conditions to further reduce sugar and fructan contents. Subsequently, the precipitate was dissolved in 50 mmollL Na-acetate buffer, pH 5, containing 0.02 % (w/v) Na-azide and centrifuged for 2 min at 10,000 Kn. The supernatant was then loaded onto a 5 mL Sephadex G-25 column previously equilibrated with 50 mmollL Na-acetate buffer, pH 5, containing 0.02 % (w/v) Na-azide. I-SST activity (1-kestose formation from sucrose) and invertase activity (fructose formation from sucrose) were measured by incubation of the desalted protein for 1 h at 30 DC in 50 mmollL Na-acetate buffer, pH 5, containing 100 or 20 mmollL sucrose and 0.02 % (w/v) Na-azide. Glucose, fructose and l-kestose were the only reaction products formed. I-FFT activity (l-kestose formation from inulin and sucrose) was measured by incubation of the desalted protein extract for 30 min at o DC in 60 mmollL MES-NaOH buffer, pH 6.25, containing 10 mmollL sucrose as fructosyl acceptor and 3 % (w/v) commercial chicory root inulin (Sigma) as fructosyl donor. I-FEH activity (fructose production from inulin) was measured by incubation of the desalted protein fraction together with 3 % (wI v) of chicory root inulin (Sigma) in 50 mmollL Na-acetate buffer, pH 5, containing 0.02 % (w/v) Na-azide at 30 DC for 2 h. In all cases reactions were stopped by heating at 95 ·C for 5 min. Samples were diluted threefold with 0.03 % (w/v) Na-azide. From

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these, 25 ILL was automatically injected onto a Dionex column (see above). For protein determination we used the method of Bradford (1976). Enzymatic activities are expressed in units (U) per g protein. One U is defined as the amount of enzyme that produced 111mol of a product in 1 min at the incubation temperature used.

Phlonn exudate Phloem exudate was collected using the EDTA-enhanced exudation technique essentially as described by Rochat and Boutin (1991). One feeder root was selected. By cutting with a razor blade the largest part of the root (about 25 em) was removed leaving only the most proximal part (5 cm) on the tap root. Afrer a shon rinsing with distilled water to remove the carbohydrates leaking out of destroyed cells, the most distal part of the remaining feeder root was placed in a tube containing 1 mL of 50 mmol/L EDTA in 10 mmol/L HEPES/KOH buffer, pH 7.5, for 15 min at room temperature. Afrer 15 min, the feeder root end was transferred to a fresh solution during a 4 h period.

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Fig. 1: Evolution of the total feeder root fresh weight during forcing of witloof chicory Ouly 10 until August 2, 1996) in a hydroponic system using four different hydroponic solutions: water (control, e), water supplemented with 0.1 mol/L NaCI (_), 0.2 mol/L NaCl (A) or 0.035 mol/L PEG-6000 (~). The last three data points of the PEG treatment are missing due to the complex interweaving of these highly branched roots (from different tap roots) under these conditions. Bars represent standard errors for n=4.

and 2 A) is invested in the production of a large number of hairy lateral roots. The importance of water economy is highlighted by the fact that fresh weight (Fig. 2A) and length of the chicon (Fig. 2 B) are negatively affected by lowering the water potential of the hydroponic solution using either NaCl or PEG. Hairy root formation is less inhibited by osmotic stress than chicon production, especially in the 0.1 mollL NaCl and PEG conditions (compare Figs. 1 and 2).

Fructans and enzymatic activities in feeder roots

Very young roots contain mainly fructose, glucose and sucrose (Table 1), probably reflecting their active metabolic state (a large percentage of the cells is still meristematic or differentiating). Small DP fructans were already apparent after the first week and their concentration increased steadily throughout the forcing period. Large DP fructans appeared later and their concentration increased concomitantly (Table Extra /UTation oftht hydroponic solution 1). It is observed that the fructose to fructan ratio greatly deTwo times 10 chicory roots were forced under the conditions de- creases as a function of time (as can be deduced from Tascribed above using the control hydroponic solution that was either ble 1). flushed with air at a rate of 500 mL min -lor not. The free oxygen Even in 75 times diluted carbohydrate extracts of the feeconcentrations in both the aerated (9.5 ppm) and non-aerated (3.3 ppm) conditions were measured using a Clark oxygen detector. der roots, fructans with a DP up to 20 could be detected afrer Afrer 3 weeks of forcing, both carbohydrate concentrations and en- 3 weeks of forcing. The pattern of these fructans (Fig. 3 A) does not correspond to the pattern in the tap roots (Fig. 3 B) zymatic activities were measured as described above. since large amounts of alternative fructans (inulo-n-oses and a second unknown series) are always detected in forced tap roots (Van den Ende et al., 1996 a, b). These alternative frucResults tans could not be detected in the young lateral roots. Afrer 3 weeks of forcing, feeder roots were divided into a Feeder root and chicon growth proximal, medial and distal part. Carbohydrate concentraThe fresh weight of the feeder roots increases (Fig. 1) to- tions in the three different parts are presented in Fig. 4. A gether with the fresh weight of the chicons (Fig. 2 A) up to comparison of enzymatic activities of the tap root and the about 3 weeks after the start of the forcing treatment. Quite three different parts of the feeder roots is presented in Fig. 5. some material (roughly 5 % of the chicon, compare Figs. 1 The highest fructose concentration and the lowest fructan

293

Chicory Fructans: Osmotic Effects

concentrations are found in the distal (youngest) part of the feeder roots (Fig. 4). This can be readily correlated with the higher invertase (and somewhat lower I-FFT activities) at that place (Fig. 5). Notice that nearly no acid invertase activity and no I-SST activity are present in the forced tap root while the I-FFT activity is ten times higher than in feeder roots and the I-FEH activity about 3 times (Fig. 5). Taken together with previous results (Van den Ende and Van Laere, 1996 b), these results indicate that high acid invertase and I-SST activities are associated with young growing material while 1-FEH or 1-FFT activities seem to increase in older tissues. Phloem exudate of a feeder root was collected using the EDTA-enhanced exudation technique. From our analysis it is clear that sucrose is the main transport carbohydrate used {Fig. 6).

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Fig. 3: A. Original chromatogram of the carbohydrates present in the feeder roots of chicory after 21 days of forcing in a hydroponic system using water (control condition). B. Original chromatogram of the carbohydrates present in the tap root of chicory after 21 days of forcing in a hydroponic system using water (control condition) . The elution positions of mannitol (M, internal reference), glucose (G), fructose (F), sucrose (S), l-kestose (I<), 1,I-nystose (N), DP5 (5) and DP6 (6) are indicated.

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Fig. 2: A. Evolution of chicon fresh weight during forcing of witloof chicory Guly 10 until August 2, 1996) in a hydroponic system using four different solutions: water (control, e), water supplemented with 0.1 mol/L NaCI (_), 0.2 mollL NaCI (A) or 0.035 mollL PEG-6000 (.6.). Bars represent standard errors for n=4. B. Evolution of the chicon length during forcing. Legend as under A.

The main effect of the 0.1 mollL NaCI treatment was a slight increase in sucrose concentration and a slighdy lower fructan concentration (data not shown). Probably the developing lateral roots can easily cope with this moderate decrease in water potential although chicon growth is already affected. In 0.2 mollL NaCl, however, not only sucrose but all fructans reach higher concentrations than in control conditions (Table 1). This coincides with an important reduction in chicon growth and feeder root fresh weight (Figs. 1 and 2). In a PEG solution with a water potential similar to the 0.2 mollL NaCl solution, there was a clear-cut increase in glucose, fructose and sucrose. Fructan concentrations on the contrary were gready reduced (Table 1). Although we did not determine the exact tissue water content or the tissue osmotic potential, the «total carbohydrate» data presented in Table 1 most probably give a good estimation of the contribution of carbohydrates to the water potential of the tissue. From this it can be concluded that carbohydrates compensate for nearly 50 % of the low water potential of the PEG solution. It is difficult to estimate the contribution in the other conditions since no data are available on Na+ and/or Cl- uptake and concentrations. The high contribution by carbohydrates (especially glucose, fructose and sucrose) in the PEG-condition is apparendy reached by limiting the polymerisation to fructan {see «mean DP» and car-

294

WIM VAN

DBN ENDB, SAM MOORS, GBRD VAN HOBNACKBR, and ANDRi VAN WRB

Table 1: Quantification of me concentrations of glucose (Glu) , frucrose (Fru) , sucrose (Sue), 1-kestose (Kes), DP4-9, DPlO-15 and

DP16-20 fructans in me developing feeder roots after 6, 16 and 23 days of forcing using water (control), 0.2 mollL NaCI or 0.035 mollL PEG-6000 as hydroponic solution. Data are expressed in flmoles carbohydrate per g fresh weight. Values are means ± SE of four determinations. Data between brackets represent percentages of rotal carbohydrate. Also the total carbohydrate concentration and me mean DP are shown. day 16

day 6

day 23

control

NaCI

PEG

control

NaCl

PEG

control

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PEG

DP10-16

19.3±2.3 (20.S) 42.4±4.5 (4S.7) 21.6±S.6 (23.3) S.O± 1.7 (S.7) 1.4±O.4 (1.6) 0

23.5±3.5 (20.9) 3S.1±4.6 (33.S) 3S.5± 12.4 (31.S) 13.S±S.7 (12.2) 1.S±0.6 (1.69) 0

45.3±7.2 (23.6) SO.6±19.6 (42.0) S6.1 ±23.4 (29.2) 9.2±1.S (4.S) 0.77±0.2 (0.4) 0

DP16-20

0

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6.9±0.S (13.1) 7.9±0.9 (1S.0) lS.6±0.6 (35.2) S.7±1.1 (16.5) 9.2±1.S (17.5) 1.3±0.3 (2.6) 0.1l±0.04 (0.22) S2.7±S.2

SO.S±7.4 (36.7) 40.S±7.7 (29.5) 33.0±4.2 (23.S) S.7±0.6 (6.39 4.6±OA (3.3) 0.46±0.09 (0.33) 0

112.7±26.S

6.5± 1.1 (1S.0) S.O± 1.2 (22.3) 9.S±0.S (27.2) S.9±0.9 (16.3) S.l ± 1.1 (14.1) 0.7±0.lS (2.0) 0.OS±0.02 (O.lS) 36.0±4.5

13S.4±20.4

4.2±0.7 (S.1) 4.3±0.6 (S.3) 13.4± 1.6 (26.1) 9.9±1.2 (19.4) lS.3±1.9 (29.S) 3.4±0.6 (6.6) 0.S9±0.OS (1.S) S1.4±6.7

4.1±0.5 (4.1) 46 . ±0.6 (4.7) 31.6±4.0 (36.6) 14.S±2.6 (17.2) 22.S±3.S (2604) 4.6±0.9 (S.3) 0.93±0.21 (1.1) S6.4± 10.2

71.S±3.9 (3S.1) S1.3±3.S (27.4) 46.2±3.9 (24.6) 1O.1±1.6 (S.4) 7.4± 1.S (3.9) 0.79±0.1 (0.42) O.06±O.OS (0.03) lS7.6±14.S

1.62±0.30

1.4±0.37

2.39±0.29

2.71±0.27

1.S2±0.21

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Total 92.S±14.2 carbohydrate Mean DP 1.47±0.22

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FI8.41 Quantification of me concentrations of glucose (glu), fructose (fru), sucrose (sue), 1-kestose (kes), 1,1-nystose (nys) and several fructan oligomers (dpn) in me proximal (black bars), medial (white bars) and distal (dashed bars) parts of the feeder roots after 21 days of forcing in a hydroponic system using water (control condition). Data are expressed in flmoles carbohydrate per g fresh weight. Bars represent standard errors for n=4.

bohydrate expressed as a percentage of total carbohydrate, Table 1). We verified that the oxygen concentration in the viscous PEG containing solution was lower than the control solution (0.7 vS. 3.3 ppm). We investigated the effect of an additional

aeration of the hydroponic solution. Similar to the findings of Albrecht et al. (1993), we found higher fructan (and sucrose) concentrations under non-aerated (hypoxic) conditions (Fig. 7). This could also be correlated with a higher I-SST activity under non-aerated conditions (Fig. 8). Therefore, the

Chicory Fructans: Osmotic Effects

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lower fructan concentrations in feeder roots growing in a PEG containing solution probably have nothing to do with hypoxia but with a real osmotic stress effect.

100

Effict ofomwlytes on core browning Some characteristics of the chicon were also scored at harvest. Lower water potentials clearly have a negative effect on productivity (Fig. 2) but the length of the central axis (even in comparison with total chicon length) is also significantly reduced (Table 2). It is demonstrated here that brown axis incidence was much greater in the conditions with lowered water availability (Table 2, Fig. 9).

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Time (min) Fig. 6: Original chromatogram of the carbohydrates present in the phloem exudate of chicory feeder roots. The elution positions of glucose (G), fructose (F) and sucrose (S) are indicated.

Discussion

Fructans have been implicated in osmoregulation and water stress (see Introduction). Therefore, we investigated here whether fructans and fructan metabolizing enzymes in the newly formed hairy roots were influenced by changing the

296

WIM VAN DEN ENDE, SAM MOORS, GUD VAN HOENACKER, and ANDRE VAN LAERE

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Fig. 7: A. Evolution of total fructan concentration in the feeder roots during forcing using aerated {O or non-aerated (_) water (control condition) as a hydroponic solution. Data are expressed in ~moles carbohydrate per g fresh weight. B. Evolution of the sucrose concentration. Legend as under A. 35

tivities that could be detected in these roots (data not shown) and still also in the distal part (i.e. the youngest, growing part) of 3-week-old feeder roots (Fig. 5). Based on the different fructan pattern obtained from the tap root and the feeder roots (Fig. 3), we suggest a real neosynthesis of inulin-type fructans (G-Fn) in the feeder roots from the sucrose that is imported from the tap root (via phloem transport, Fig. 6). Regarding fructan concentrations in growing feeder roots, we found strongly different effects by inclusion of 0.2 moUL NaCI or the iso-osmotic PEG in the bathing solution (Table 1). The higher fructan concentrations obtained with the 0.2 mollL NaCl treatment might be explained by uptake of

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FII.8: Activities of invertase, I-SST, I-FFT and I-FEH in chicory

feeder roots forced in the control hydroponic solution without (dark bars) or with (white bars) extra aeration of the hydroponic solution.

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Table 2: Effect of osmolytes on axis length and internal browning incidence in witloof chicons (n = S) after 23 days of forcing.

axis length cm % of chicon

Internal browning

Control

0.1 mol/L NaCl

0.2 mol/L NaCl

7.7±0.3 60.3± 1.5

4.S±OA 49.S±3.6

2.9±0.1 37.0±0.S

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water potential of the bathing solution during the forcing process. As a fuction of feeder root development the concentration of monosaccharides (especially fructose) strongly decreases while fructan concentrations strongly increase (Table I). The high concentration of fructose (and glucose) in the young roots probably arises from the very high invertase ac-

Fig. 9: Photograph showing the internal axis of the chicons obtained after 23 days of forcing under four different conditions. From the lefr to the right: control condition, 0.1 + 0.2mol/L NaCl condition and 0.035 moUL PEG condition.

Chicory Fructans: Osmotic Effects

297

Na + (and! or Cn with a concomitant alleviation of the water non-permeant osmoticum PEG in the bathing solution stress applied. Alternatively, the toxic effects of the large ion largely effected yield and quality of the chicons and resulted concentrations in the feeder roots could be the reason for the in higher mono- and dissacharide concentrations and lower reduced growth, allowing fructans to accumulate to higher fructan concentrations in the feeder roots. levels under these conditions. In developing chicory feeder roots, PEG-induced osmotic Acknowledgements stress -does not induce but rather prevents an accumulation of The authors would like to thank E. Nackaerts and P. Janssens for fructans while much higher fructose, glucose and sucrose valuable technical assistance. The help of G. Claessens, J. de Greeff concentrations are found (Table 1). These data are in con- and R van Rossum (Proefbedrijf voor witloof, Herent, Belgium) is tradiction with the observation that PEG-induced drought also highly appreciated. W. Van den Ende is also grateful to the Nastress resulted in a 7-fold increase in bacterial levan-type fruc- tional Fund for Scientific Research (Belgium) for giving a grant for tan concentration in leaves of tobacco transformed with the research assistants. SacB gene from Bacillus subtilis fused to the carboxypeptidase Y vacuolar sorting signal and placed under control of the References constitutive Ca MV 35S promotor (cpySacB plants, PilonSmits et al., 1995). However, the amount of levan accumulated in the leaves was much lower than the concentrations of ALBRECHT, G., S. KAMMERER, W. PRAZNIK, and E. M. WIEDENROTH: Fructan content of wheat seedlings under hypoxia and folglucose, sucrose and especially fructose, which were also inlowing re-aeration. New Phytol. 123,471-476 (1993). creased by drought stress. BIELESKI, S.: Fructan hydrolysis drives petal expansion in the Our results showing a great increase in mono- and disacephemeral daylily flower. Plant Physiol. 103,213-219 (1993). charide concentrations under drought stress are similar to the BRADFORD, M. M.: A rapid and sensitive method for the quantitafindings of Spollen and Nelson (1994) working on the leaves tion of microgram -quantities of protein utilizing the principle of of tall fescue (Festuca arundinacea Schreb.). However, these protein-dye binding. Anal. Biochem. 72,248-254 (1976). results can be reached by two different processes: an inhibi- CHATTERTON, N. J., P. A. HAIuSSON, W. R THORNLEY, and J. H. BENETT: Separation and quantification of fructan (inulin) oligotion of fructan synthesis or an enhanced breakdown of frucmers by anion exchange chromatography. In: Inulin and Inulintans. Chicory feeder roots are initiated in the osmotic solucontaining Crops (FUCHS, A. ed.), pp. 93-99. Elsevier, Amstertion. Therefore, the stress condition is likely present before dam (1993). any fructan is present in the feeder root tissue. Indeed, so far CLAESSENS, G., A. VAN WRE, and M. DE PROFT: Purification and there is no experimental support to believe in a direct transfer properties of an inulinase from chicory roots. J. Plant Physiol. of fructan from the tap root to the growing feeder roots. 136, 35-39 (1990). Therefore, fructan chain length and concentration in feeder DEN OUTER, R w.: Internal browning of witloof chicory (Cichoroots are likely controlled by fructan synthesizing and degradrium intybus L.). J. Hort. Sci. 64,697-704 (1989). ing activities within feeder roots. Since we did not have EDELMAN, J. and T. G. JEFFORD: The mechanism of fructan metabolism in higher plants as exemplified in Helianthus tuberosus. New enough material to measure enzymatic activities as a function Phytol. 67, 517-531 (1968). of feeder root development under all of the different forcing conditions we used, conclusions about fructan concentrations HENDRY, G.: Evolutionary origins and natural functions of fructans. A climatological, biogeographic and mechanistic appraisal. New being mainly controlled by synthesis or hydrolysis are premaPhytol. 123,3-14 (1993). ture at this time. In the case of tall fescue, fructans were al- Koops, A. J. and H. H. JONKER: Purification and characterization of ready present before drought stress was induced and the stress the enzymes of fructan biosynthesis in tubers of Helianthus tubeconditions resulted in a depolymerization of fructan by frucrosus Colombia. II. Purification of sucrose: sucrose 1-fructosyltan hydrolases, yielding high concentrations of osmotically transferase and reconstitution of fructan synthesis in vitro with purified SST and FFT. Plant Physiol. 110, 1167-1175 (1996). active hexose and sucrose. This depolymerization was also demonstrated in daylily petals when the flower opens (Bie- LIMAMI, A. and T. LAMAzE: Calcium (45 Ca) accumulation and transport in chicory (Cichorium intybus L.) root during bud developleski, 1993). ment (forcing). Plant and Soil 138, 115-121 (1991). Both addition of NaCl or PEG in the hydroponic solution LUSCHER, M., c. ERDIN, N. SPRENGER, U. HOCHSTRASSER, T. BOLnot only resulted in a drastic decrease in chicon length and LER, and A. WIEMKEN: Inulin synthesis by a combination of puriweight (Fig. 2), but also in a tremendous increase of internal fied fructosyltransferases from the tubers of Helianthus tuberosus. browning in the heads of witloof chicory (Table 2, Fig. 9), FEBS letters 385, 39-42 (1996). the most important disorder (especially occurring in hydro- MONEY, N. P.: Osmotic pressure of aqueous polyethylene glycols. ponics) considerably reducing the market value of the chicon. Relationship between molecular weight and vapour pressure deficit. Plant Physiol. 91, 766-769 (1989). We suggest that the reduced tran~iration stream under these conditions might decrease the Ca + import in the developing PIWN-SMITS, E. A. H., M. J. M. EBSKAMP, M. J. W. JEUKEN, P. J. WEISBEEK, and S. C. M. SMEEKENS: Improved performance of buds and this in turn might aggravate this physiological distransgenic fructan-accumulating tobacco under drought stress. order (Den Outer, 1989; Limami and Lamaze, 1991). Conclusion

An inulin series of fructans is produced in developing feeder roots during forcing of witloof chicory. Inclusion of a

Plant Physiol. 107, 125-130 (1995). ROCHAT, C. and J.-P. BOUTIN: Metabolism of phloem-borne amino acids in maternal tissues of fruit of nodulated or nitrate-fed pea plants (Pisum sativum L.). J. Exp. Bot. 42,207-214 (1991). SPOLLEN, W. G. and C. J. NELSON: Response of fructan to water deficit in growing leaves of tall fescue. Plant Physiol. 106, 329-336 (1994).

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J. DE RooVBR, and A. VAN WIlE: In vitro synthesis of fructofuranosyl-only oligosaccharides from inulin and fructose by purified chicory root fructan:fructan fructosyl transferase. Physiol. Plant. 97, 346-352 (1996a). VAN DEN ENDS, A. MINTISNS, H. SPBLBBRS, A. A. ONUOHA, and A. VAN WIlE: The metabolism of fructans in roots of Cichorium intybusduring growth, storage and forcing. New Phytol. 132,555563 (1996b). VAN DEN ENDE, W. and A. VAN WIlE: Dt novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes VAN DEN ENDS, W.,

and ANDRE VAN WIlE (sucrose: sucrose fructosyl transferase and fructan: fructan fructosyl transferase) from chicory roots (Cichorium intybus L.). Planta 200,335-342 (1996a). - - Fructan synthesizing and degrading activities in chicory roots (Cichorium intybus L.) during fidd-growth, storage and forcing. J. Plant Physiol. 149,43-50 (1996b). VAN DEN ENDE, w., D. VAN WONTERGHEM, P. VERHAERT, E. DEWIL, A. DE LooF, and A. VAN WIlE: Purification and characterization of fructan: fructan fructosyl transferase from chicory roots (Cichorium intybus L.). Planta 199,493-502 (1996c).