Synthesis of Inulin Oligomers in Tissue Slices, Protoplasts and Intact Vacuoles of Jerusalem Artichoke

J. Plant Physiol. Vol. 138. pp. 204 - 210 (1991)

Synthesis of Inulin Oligomers in Tissue Slices, Protoplasts and Intact Vacuoles of Jerusalem Artichoke NICHOLAS

C. CARPITA 1, FELIX KELLER2, DAVID M.

1

GIBEAUT , THOMAS

L.

HOUSLEy3,

and

PHILIPPE MATILE2 1 2

3

Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907 (U.S.A.) Institute of Plant Biology, University of Zurich, Zollikerstr. 107, CH-8008 Zurich, Switzerland Department of Agronomy, Purdue University, West Lafayette, Indiana 47907 (U.S.A.)

Received June 25, 1990 . Accepted January 10, 1991

Summary Tissue slices and protoplasts of Jerusalem artichoke (Helianthus tuberosus L. cv. Bianco) incorporate radioactivity from glucose into sucrose, 1-kestose, nystose (inulin tetrasaccharide) and other fructan oligomers, and intact isolated vacuoles absorb sucrose and incorporate radioactivity from sucrose into 1kestose. Gas-liquid chromatography and radiogas proportional counting demonstrated that, in all cases, the glucosyl and internal fructosyl units of 1-kestose contained substantial amounts of radioactivity whereas the terminal fructosyl unit was essentially devoid of label. We propose that incorporation of radioactivity into 1-kestose is primarily from transfer of the unlabeled terminal fructosyl unit of nascent 1kestose to labeled sucrose by fructan : fructan fructosyl transferase and not by sucrose: sucrose fructosyl transferase. Such reactions do not result in a net gain in fructan, and hence, more definitive estimations of SST activity and the net synthesis of fructans in vivo are needed. Our success preserving fructan metabolism in isolated vacuoles, however, provides a system to examine more closely the role of the tonoplast in vectorial transport of sucrose into the vacuole, glucose and fructose out of the vacuole, and the association of these transport processes with net increases in synthesis of 1-kestose and fructans.

Key words: Helianthus tuberosus, protoplasts, vacuoles, inulin, sucrose, l·kestose, carbohydrate metabolism, sucrose: sucrose Jructosyl transferase, Jructan :Jructan Jructosyl transferase, gas chromatography-radiogas pro· portional counting. Abbreviations: FFT = fructan: fructan fructosyltransferase; LSS = liquid scintillation spectroscopy; SST = sucrose: sucrose fructosyltransferase; TF A = trifluoroacetic acid.

Introduction Fructans, polymers of fructofuranosyl units initiated with sucrose, are the principal storage forms of carbohydrate in many plant species (Hendry, 1987; Meier and Reid, 1982; Pollock and Chatterton, 1988; Wiemken et aI., 1986). Fructans are temporary storage reserves involved in diurnal translocation of photosynthate from leaves of cool-season grasses (Gordon, 1986), longer term storage in grass stems and inflorescences for eventual transfer to developing grains (Hendrix et al., 1986; Pollock and Chatterton, 1988), and even seasonal © 1991 by Gustav Fischer Verlag, Stuttgart

storage in underground storage organs of Jerusalem artichoke, dahlia, and chicory (Edelman and J efford, 1968). The structures of higher plant fructans are diverse but generally fall into four categories: the (2-+1}-linked {3-o-fructans (inulins) such as those found in Jerusalem artichoke and dahlia tubers, the (2-+6~linked {3-o-fructans (phleins) of some grasses such as Phleum pratense and Festuca arundinaceae, the highly branched {2-+1} and (2-+6) mixed-linkage ,B-o-fructans and fructan oligomers of other grasses such as Triticum aestivum and Hordeum vulgare, and the neokestose series of (2-+1}-linked ,B-o-fructans such as those of Aspa-

Synthesis of Inulin Oligomers in Tissue Slices, Protoplasts and Intact Vacuoles of Jerusalem Artichoke

ragus and Allium that elongate from both the glucosyl and fructosyl unit of the initial sucrose (Carpita et aI., 1989; Meier and Reid, 1982; Shiomi, 1989). Despite such broad differences in structure, it is widely accepted that 1-kestose, iJ-D-fructosyl-(2-+ 1)iJ-D-fructosyl(2+--+1)a-D-glucose, is the principal starting molecule for the synthesis of all fructans, and 6-kestose and neokestose are trisaccharides formed by certain species as additional substrates. The synthesis of 1-kestose from two molecules of sucrose is catalyzed by the dis mutase sucrose: sucrose fructosyl transferase (SST; E.C. 2.4.1.99), established by Edelmann and Jefford (1968) in the pathway of inulin biosynthesis in Jerusalem artichoke. Because fructose is not a product of the SST reaction, the enzyme is not an invertase (Wiemken et aI., 1986). Formation of 1-kestose is virtually irreversible since the free energy of sucrose hydrolysis is so high (~G = 27.6 kJ mol-I) compared to that of the fructosyl linkages (16.8 kJ mol-I) (Lewis, 1984). The early models developed by Edelman and Jefford (1968) predicted that SST was a cytosolic enzyme whereas the fructan: fructan fructosyl transferase (FFT; E.C. 2.4.1.100) involved in chain elongation was on the tonoplast, fulfilling the dual role of transfer of the terminal fructosyl unit of 1-kestose across the tonoplast and formation of fructans in the vacuole. The first step in chain elongation is the transfer of a terminal fructosyl unit from 1-kestose to the 0-1 of the terminal fructose of another 1-kestose to form the inulin tetrasaccharide, nystose (Meier and Reid, 1982). The transport model of Edelman and Jefford (1968) went essentially untested for over fifteen years until Wagner et ai. (1983) and Frehner et ai. (1984) showed convincingly in barley and Jerusalem artichoke that fructans and all enzymes of fructan synthesis, including SST, were in the vacuole. Because fructan exohydrolase is also located in vacuoles, the initiation and synthesis of fructans and their subsequent hydrolysis are confined to one compartment (Frehner et aI., 1984). Lysis of vacuoles renders SST soluble (Frehner et aI., 1984; Darwen and John, 1989). It is likely that synthesis and degradation is regulated temporally rather than by partitioning of enzyme and substrate. Our recent observations on the synthesis of fructans in vivo in wheat leaf blades shed some light on this problem. Leaf blades induced to form fructans and pulsed in 14C02 accumulated radioactivity rapidly into 1-kestose. By derivatization of extracted 1kestose and separation by gas-liquid chromatography, we were able to determine the specific activity of each of the glycosyl units. Substantial amounts of radioactivity accumulate in glucose and the internal fructosyl unit, but the terminal fructosyl unit initially contained much lower amounts of radioactivity (Kanabus et aI., 1991). With longer incubations in 14C02, the specific activity of the terminal units slowly approached values of the other glycosyl units. Dickerson and Edelman (1966) observed similar labeling kinetics of 1kestose in Jerusalem artichoke tubers after pulse-labeling leaves with 14C02. These reaction kinetics indicate that radioactive sucrose was translocated or formed quickly in the cytosol, and terminal fructosyl units were added from a vacuolar pool of relatively unlabeled sucrose. The enzyme SST is a dismutase and, hence, cannot differentiate identical substrates in the same compartment. We considered two pos-

205

sible explanations for such kinetics. First, SST may be in tight association with the tonoplast-localized sucrose transporter and can discriminate between the two pools of sucrose, catalyzing the transfer of the fructosyl unit specifically from the relatively unlabeled sucrose of the vacuole to the incoming labeled sucrose from the cytosol. Second, FFT, rather than SST, may transfer a relatively unlabeled fructose from vacuolar 1-kestose to labeled sucrose transported into the vacuole from the cytosol. Edelman and Dickerson (1966) showed that such transfer from 1-kestose to sucrose was possible in vitro with extracts of Jerusalem artichoke tuber. The developmentally regulated synthesis of inulins in tubers of Jerusalem artichoke results in accumulation of 80 % of the dry weight in fructan, and this system is an obvious convenient model to study fructan synthesis. We examined the short-term synthesis of 1-kestose in vivo from 14C-D_ glucose in tissue slices and protoplasts of the developing tubers to determine if the asymmetric use of sucrose by SST or FFT in a seasonal storage tissue was homologous to the diurnal storage in wheat leaf blades. We then isolated intact vacuoles from protoplasts of the developing tubers and examined formation of fructan oligomers from 14C-sucrose in vitro as a first step toward preservation of the synthesis of 1kestose.

Materials and Methods Preparation of Tissue Sections Tubers of Jerusalem artichoke (Helianthus tuberosus L., cv. Bianco) were harvested during early development from plants grown in a nursery garden of the Institute for Plant Biology, University of Zurich. Tubers, 15 to 30 g, were excised from the plants and washed with cold tap water, and the thin, brown epidermis was peeled away from the crisp, white cortical tissue. The tissues were immediately soaked in a Wash Medium (25 mM Mes-Tris, pH 5.5, 10 mM Tris-ascorbate, and 1 mM CaCh) supplemented with 0.2 M glycine betaine. Flat slices of tissue 1 to 2 mm thick were shaved from the tubers with a «truffle cutten>, a stainless-steel plate containing an embedded blade and a thumb screw to adjust height. These slices were collected in additional Wash Medium. Disks 14 mm in diameter were excised from the tissue slices with a cork borer and floated in fresh Wash Medium supplemented with 0.8M glycine betaine. Preliminary experiments showed that accumulation of glucose and subsequent formation of sucrose and fructan oligomers was stimulated markedly by incubations in medium of such high concentrations of osmoticum. Three labeling experiments were performed with samples taken intermittantly from 6 min to 4 h. The amounts of radioactivity were scaled up 10-fold in the experiments shown here because of extensive chemical characterization of the various products that followed.

Synthesis of Labeled Sucrose and Fructan Oligomers in Tissue Slices When enough disks had been collected, the Wash Medium was changed, and about 50 disks were incubated in 20 mL of medium with gentle shaking at 24°C for 1 h in a 90-mm diameter Petri dish 15 mm deep. In some experiments, 100 mL of Wash Medium containing 135/LCi[U-1 4 C]-D-glucose (295mCi/mmol, CEA, Gif-surYvette, France) were then added, and samples containing 10 to 14 disks were removed intermittantly between 30 min and 4 h of labeling. In other experiments, 5 to 7 disks in 10 mL of medium con-

206

NICHOLAS C. CAIlPITA, FELIX KELLER, DAVID M. GIBEAUT, THOMAS L. HOUSLEY, and PHILIPPE MATILE

taining 40 iLCi or 135 iLCi [U-1 4 C]-D-glucose were incubated 6 to 30 min. Values reported here are the mean of samples from two separate experiments. After incubations, the disks were blotted quickly on paper towels to remove excess medium and gently swirled for 10 to 20s in fresh unlabeled Wash Medium twice with blotting between each wash. After a final blotting, the disks were transferred to the barrel of a tared 5-mL disposable syringe fitted with a single GFI F glass fiber filter mat, fresh wt of the sample was determined, and the assembly was dipped into liquid N2. The syringe barrel containing the frozen disks was fitted to a 1.5-mL Eppendorf centrifuge tube, and, after thawing, the assembly was spun at 3500 x g for 20 min in a refrigerated centrifuge to express most of the sap from the thawed disks into an Eppendorf tube. The expressed sap (ca. 250 iLL) was diluted with an equal volume of water and added to a spin-filter assembly (Schleicher and Schuell, Feldbach, Switzerland) containing 200 iLL packed-volume each of Serdolit Red Micro (H + form) and Serdolit Blue Micro (HC0 3 - form) 50-100-iLm ion exchange resins (Serva Biochemicals, Heidelberg, FRG) and incubated with the exchangers for at least 10 min. The filter assembly containing a 0.45 iLm pore nylon membrane was fitted to a 1.5-mL Eppendorf tube and centrifuged at 400 x g for 15 min. The clear, colorless, deionized sap (ca. 450 iLL) was mixed with 1.0 mL of acetonitrile to precipitate higher molecular weight fructans. The samples were microfuged for 4 min, and the supernatant was transferred to a new tube, concentrated under a stream of N2 at 45°C, and adjusted with water to 500 iLL for further analysis.

Preparation ofProtoplasts and Vacuoles Thin tissue slices made by the truffle cutter in Wash Medium containing 1 M sorbitol were diced further into pieces about 2 to 10mm 3, washed with additional sorbitol Wash Medium, and transferred to sorbitol Wash Medium containing 0.2 % pectolyase Y-23 (Seishin Pharmaceutical, Tokyo, Japan), 1 % bovine serum albumin, and 1 % cellulase CELF (Worthington Biochemicals, Irvine, CA) or 2 % cellulase Y-C (Seishin Pharmaceutical; Digestion Medium). The tissue pieces were incubated for 15 h at 30°C with gentle gyratory shaking. The protoplasts were filtered free of undigested tissue pieces though cotton cloth into Protoplast Medium (1 M glycine betaine, 5 mM Mes-Tris, pH 5.5, and 1 mM CaCh). The fructan-Iaden protoplasts settled without centrifugation within 5 min from 35 mL of Protoplast Medium in a 40-mL conical tube. The medium was aspirated from above the settled protoplasts, and the protoplasts were suspended in fresh medium and allowed to settle three more times to ensure adequate removal of the Digestion Medium. The settled protoplasts were dispensed into 250-iLL aliquots into the tip of 40mL conical tubes and used directly for labeling studies. For preparation of vacuoles, washed protoplasts were transferred to 10 volumes of Lysis Medium (0.8M glycine betaine, 40mM Hepes-KOH, pH 8.4, and up to 25 mM EGT A). Protoplasts in 20mL of Lysis Medium were gently swirled in a 100-mL Erlenmeyer flask warmed to about 35°C. Progress of lysis was noted subjectively by appearance of coagulated cytoplasm and analytically by microscopy. When substantial lysis was observed (ca. 10 min), the heavy vacuoles and unlysed protoplasts were washed three times by settling (as described for protoplasts) in 35mL each of Vacuole Medium (1 M glycine betaine, 20 mM Hepes, KOH, pH 7.6, 2mMEGTA, 2mMCaCh, 2mMMgCh, and 3mMKCI). Yield and purity of vacuoles was determined by comparison of malate dehydrogenase (cytosolic marker) and N-acetyl glucosaminidase (a vacuole marker) as described by Frehner et al. (1984). The vacuoles (containing some unlysed protoplasts) were transferred in 250-iLL aliquots into 40-mL conical tubes for labeling studies. Reactions were begun by addition of 100 iLL, or 1.0 mL in some cases, of Protoplast Medium containing 10 or 20 iLCi of [U_ 14C]-D_

glucose (295mCi/mmol) or [U-1 4 C]-sucrose (540mCi/mmol; CEA) to the protoplast preparations or the same amount of labeled sugar in Vacuole Medium to the vacuole preparations. After pulse labeling periods that ranged from 10 to 30 min for protoplasts and 5 to 20 min for vacuoles, the preparations were diluted with 35 mL of appropriate unlabeled medium, centrifuged for 1 min at 100 x g, and the loose pellets were collected by a wide-bore Pasteur pipet and frozen in liquid N2 in 1.5-mL Eppendorf centrifuge tubes. Experiments were performed twice with essentially the same results, the latter experiment with 2-fold higher amounts of label are reported here.

Extraction of sugars from protoplasts and vacuoles Water (250 iLL) was added to the frozen protoplast and vacuole preparations, and the mixtures were incubated in a sonic bath at ambient temperature for 3 min to burst membrane vesicles. The suspensions were added directly to the ion exchange resins in spincolumns, and a deionized solution was prepared as described for the preparations from tissue slices. One mL of acetonitrile was added to the 450 iLL solution recovered from the ion-exchange spin column in Eppendorf centrifuge tubes to precipitate the high molecular weight fructans. The suspensions were microfuged for 4 min, and the supernatants were transferred to new tubes. The pellets were dissolved in 300 iLL of water by gentle agitation in a sonic bath at ambient temperature, reprecipitated by addition of 700 iLL of acetonitrile, and microfuged again. The supernatants were combined and concentrated by evaporation at 45°C in a stream of N2 and adjusted to 500 iLL with water. Ten to 50 iLL were assayed for radioactivity by liquid scintillation spectroscopy (LSS), and the remainder were saved for HPLC analysis and collection. The pellets were dissolved in 1.0 mL of water, and 100-iLL samples were assayed for radioactivity by LSS.

Analysis ofRadioactive Sucrose and Fructan Oligomers The sugars were separated by HPLC on a 125-mm x 4.6-mm Permacoat NH2 (3-iLm) analytical column (Stagroma, Wallisellen, Switzerland) isocratically in 70 % acetonitrile in water at 0.8 mLI min at ambient temperature. The oligomers were detected by refractometry and quantified with a computing integrator. Standard solutions for quantitation were fructose, glucose, sucrose, and «neosugar» containing fructose, glucose, 1-kestose, nystose, and inulin pentasaccharide in known proportions (BioScience Laboratories, Meija Seika Kaisha Ltd., Kawasaki, Japan). Radioactivity was determined in independent runs with a Radiomatic A-250 Flow-One/Beta radioactivity flow detector (Canberra Packard, Zurich, Switzerland) with post-column addition of Pico-Aqua liquid scintillation cocktail (Canberra Packard) at 1.0 mL/min. Radioactive sucrose, 1-kestose, and nystose were then collected on the same column isocratically in 73 % acetonitrile (to increase separation of oligomers) at 0.8 mLimin with monitoring by refractometry. The acetonitrile was evaporated at 45°C in a stream of N 2. The dry samples were dissolved in 2.0 mL of water, and 100 iLL was taken for determination of radioactivity by LSS and 100 iLL was taken for determination of fructose by a furanose-specific anthrone assay Oermyn, 1956). The remainder was frozen and lyophilized in 15-mL glass tubes for methylation analyses.

Methylation Analysis of Sugars by Gas Chromatography·Radio Gas Proportional Counting Partially methylated alditol acetate derivatives of sucrose and the fructan oligomers were prepared as described previously (Carpita et al., 1989; Carpita and Shea, 1989) except that hydrolysis of the permethylated derivatives was in 1.0mL of 1MTFA (containing 5nCi and 50 nmol p4C]-myo-inositol and internal standard) at 70°C for

Synthesis of Inulin Oligomers in Tissue Slices, Protoplasts and Intact Vacuoles of Jerusalem Artichoke 30 min. The TFA was evaporated at 40°C in a stream of N2 after addition of 1.0 mL of tert-butyl alcohol to reduce decomposition of the fructofuranosyl units. The derivatives were separated in a 0.75mm x 30-m wide-bore glass capillary column of SP-2330 (Supelco, Bellafonte, Pennsylvania, USA) temperature programmed from 160°C to 210 °C at 2 °C/min then to 240°C at 5 °C/min with a 10min hold at the upper temperature. Derivatives were quantified by flame ionization detection after post-column effluent splitting, with radioactivity determined in 95 % of the total effluent diverted to a Model 894 radiogas proportional counter (Packard Instruments, Downer's Grove, Illinois, USA) connected to the GC as described (Shea et al., 1989).

8

A

sucrose o·

/

0

f: c

:::t

~ Ci

Il.

~"-

II

Results and Discussion

Uptake and Incorporation of Labeled Sugars into Sucrose, l-Kestose, and Nystose Tissue slices rapidly absorbed [U-14C]-D-glucose from the incubation medium and incorporated the labeled glucose into sucrose, l-kestose, nystose, and trace amounts of higher

A 9

7

/"

"

10

d

l-kestose •

//0 . "_

'0 E 4

B

207

07

•

[ ~

5

'0 E

:::t

~

Il.

Ci

nystose

-----0 20 10 30 Inc ubation Time, h

Incubation Time, min

Fig.2: Accumulation of radioactivity from [U)4C}D-glucose into sucrose, l-kestose, and nystose in Jerusalem artichoke tissue slices and protoplasts. The three sugars separated by HPLC (Fig. 1) were detected by refractometry for collection, and specific activity was determined in each sample by liquid scintillation spectroscopy and fructose was determined by a furanose-specific anthrone assay Oermyn, 1956). A. Tissue slices. B. Protoplasts.

2

6

3

-

0

inulin oligomers (Fig. 1 A). Rates of incorporation were

;,.

,~iG- 5

6

B

2

I

6 ~

N

I

S

C ~

0-

3

0

A5

0

18 r-

C

2

3

12

6

o o

I

IV 5

5 10

15

Run Time, min

Fig. 1: Incorporation of radioactivity in sucrose, l-kestose, and fructan oligomers of tissue slices and protoplasts given [U)4C}oglucose and vacuoles given [U)4C}sucrose. Sugars in deionized extracts were separated isocratically in 70 % acetonitrile on a NH2 column (3/Lm) and radioactivity was determined by flow detection after post-column addition scintillation cocktail. Peaks identified by co-elution with standards are: 1) fructose + glucose, 2) sucrose, 3) 1kestose, 4) [unknown], 5) nystose, and 6) inulin pentasaccharide. A. Extract from tissue slices after incubation 4 h in [U)4C-}-D-glucose. B. Extract from protoplasts after incubation 30 min in [U- '4 C}oglucose. C. Extract from vacuoles after incubation 20 min in rU14C}sucrose.

nearly linear for about 2 h, then decreased, perhaps because of saturation of the cytoplasmic pools of sugar with the label (Fig. 2 A). Endogenous pools of sucrose and l-kestose are quite large in tissue slices, and the concentrations do not decrease substantially during the incubation up to 4 h. Protoplasts recovered after 15 h in Digestion Medium still contain about 20 mM sucrose and 10 mM l-kestose, about 60 % and 50%, respectively, of their original concentrations in fresh tissue slices. Radioactivity from 14C-D-glucose accumulated within minutes in both sucrose and l-kestose in isolated protoplasts, indicating that enzymes of sucrose and fructan synthesis are still operating in protoplasts, but synthesis of nystose and higher oligomers is lower relative to rates of sucrose and l-kestose formation in tissue slices (Fig. 1 B). Because of shorter labeling times, incorporation of label into sucrose and l-kestose was essentially linear in protoplasts (Fig. 2 B). Vacuoles isolated from protoplasts constituted the in vitro synthesis system. These vacuoles also accumulated substantial amounts of radioactive sucrose, and much of the radioactivity was incorporated into l-kestose (Fig. 1 C). Very little radioactivity was incorporated into nystose in these short incubations. Increases in specific activity was parallel in sucrose and l-kestose (Fig. 3). Because the vacuole preparation was contaminated with about 20 % protoplasts (based on relative activities of cytoplasmic and vacuolar markers), the incorporation of 14C-D-glucose and 14C-sucrose was compared in vacuole and protoplast preparations. Protoplasts formed much less labeled l-kestose from 14C-sucrose than did isolated vacuoles (Table 1). Neither sucrose nor l-kestose could be made by vacuoles when labeled glucose was provided. Hence, the contaminating protoplasts were apparently damaged or leaky so that the ability to form sucrose was im-

208

NICHOLAS C. CARPITA, FELIX KELLER, DAVID M. GIBEAUT, THOMAS L. HOUSLEY, and PHILIPPE MATILE A

20

B

I

o

[ "0 S

::i

-...

f-Glc

t-Fru

~

10

I-Fru

t-Glc

\-kestose

~

0...

Q

/-Fru

nystose

A-

I-Glc

1-Fru

t-Fru

(-Glc

l-Fru

20 Incubation Time, min Fig. 3: Accumulation of radioactive [U)4C]-sucrose and incorporation of radioactivity into 1-kestose and nystose in isolated vacuoles. Sugars were separated and specific activities determined as described in Fig. 2. Table 1: Comparison of the formation of 1-kestose from [U- 14C]-Dglucose or [U)4C]-sucrose by vacuoles and protoplasts.

a

b

c

d

Substrate

Protoplastsa

[U- 14C]-sucrose c [U)4C]-D-glucose d

42,200 46,700

Vacuoles b

dpmlJtmole

173,800 tr

Protoplasts were generated from tissue pieces incubated 15 h in Worthington CELF cellulase and Onozuka pectinase Y-23, recovered by sedimentation at 1 g, and washed three times and suspended in 1 M glycine betaine,S mM Mes-Tris, pH 5.5, and 1 mM CaCho Vacuoles were prepared by suspension of the protoplasts in 0.8 M glycine betaine, 40 mM Hepes-KOH, pH 8.4, and 20 mM EGTA with warming to 35°C for about 10 min. Intact vacuoles and unlysed protoplasts settled at 1 g were washed twice and suspended in 1 M glycine betaine, 20 mM Hepes, KOH, pH 7.6, 2 mM EGTA, 2 mM CaCh, 2 mM MgCh, and 3 mM KC!. Aliquots {250 JtL} of settled protoplasts and vacuoles were incubated with 10 JtCi [U- 14C]-sucrose {540 mCi/mmol; CEA} for 15 min at 25°C, diluted IsO-fold with unlabeled Protoplast or Vacuole Medium, and protoplasts and vacuoles were collected after centrifugation 1 min at 100g. Sugars in deionized extracts were separated by HPLC {Fig. 1} and specific activities of 1kestose were calculated after determination of radioactivity by liquid scintillation spectroscopy and fructose according to Jermyn {1956}. Aliquots {250 JtL} of settled protoplasts and vacuoles were incubated with 20 JtCi of [U)4C]-D-glucose {295 mCi/mmol} for 20 min at 25°C, and prepared as described for sucrose-labeled materia!'

paired. Further, these data indicated that sucrose uptake actually may be from damaged protoplasts in the preparation

Temperature Programs

Fig. 4: Determination of radioactivity in individual glucosyl and fructosyl units of sucrose and 1-kestose from tissue slices given [U14C]-D-glucose and vacuoles given [U)4C]-sucrose. Partially methylated alditol acetate derivatives were made of sugars collected from HPLC {Fig.1) and separated by gas-liquid chromatography. Specific activities were quantified by digital integration of both mass by flame ionization detection {lower traces} and radioactivity by radiogas proportional counting {upper traces}. A. Sucrose from tissue slices incubated 4h in [U)4C]-D-glucose. B. Sucrose from vacuoles incubated 20 min in [U)4C]-sucrose. C. 1-Kestose from tissue slices incubated 4 h in [U)4C]-D-glucose. D. 1-Kestose from vacuoles incubated 20 min in [U)4C]-sucrose. Peaks identified by co-elution with standard derivatives verified by gas chromatography-electron impact mass spectrometry are: t-Fru, non-reducing terminal fructosyl unit; t-Glc, non-reducing terminal glucosyl unit; and 1-Fru, {2-1}-linked fructosyl unit. Skewed peak shape is a result of slight overloading necessary to achieve accurate determination of both mass and radioactivity.

and only vacuoles were accumulating substantial amounts of radioactivity from labeled sucrose to form l-kestose in vitro. Analysis a/Specific Activities o/Glycosyl Moieties a/Sucrose and l-Kestose

After separation of partially methylated alditol acetate derivatives by gas-liquid chromatography, the amounts of radioactivity in each glycosyl unit was assayed by radiogas proportional counting (Fig. 4 A - D). The sucrose fraction from HPLC (Fig. 1 A) contained an additional peak containing radioactivity (Fig. 4 A), but we have not yet identified the substance. Amounts of radioactivity in the terminal fructosyl moiety of sucrose from tissue slices was much lower than that in the terminal glucosyl unit (Fig. 4 A), whereas the fructosyl and glucosyl moieties of sucrose accumulated by vacuoles contained nearly equal amounts of label (Fig. 4 B). In

Synthesis of Inulin Oligomers in Tissue Slices, Protoplasts and Intact Vacuoles of Jerusalem Artichoke Fig. 5: Changes in specific activities of fruc· tosyl units relative to glucosyl units of ra· dioactive sucrose and l-kestose during incubations of tissue slices and protoplasts in [U-,4C]-D-glucose or vacuoles in [U-'4C]sucrose. Sucrose and l-kestose extracted from tissue slices and protoplasts given [U14C}o-glucose and vacuoles given [U-'4C]sucrose were separated by HPLC (Figs. 2 and 3) and specific activities in glycosyl moieties calculated from digital integration of signals from flame ionization detection and radiogas proportional counting (Fig. 4). Changes in specific activities of the terminal fructosyl unit of sucrose (.) and I-linked (internal) fructosyl unit of l-kestose (0 ) were calculated relative to the specific activity of the terminal glucosyl unit. A. Tissue slices. B. Protoplasts. C. Vacuoles.

B

C

10

l-

f-

0.8

l-

I-

A

,

'0

0 0

'.

0.6 l·keSlose

0.4

'~ .

0.2

'"

2

Incubation Time, h

tissue slices given 14C-o-glucose and vacuoles given 14Csucrose, no radioactivity was found in the terminal fructosyl unit of l-kestose, whereas the glucosyl unit and I-linked fructosyl unit contained substantial amounts of label (Fig. 4 C and D). The terminal fructosyl unit of l-kestose from protoplasts was also devoid of label (not shown). From quantitation of mass and radioactivity simultaneously, the specific activities of the glycosyl units were calculated. In tissue slices and protoplasts, the specific activities of both glycosyl units of sucrose and the glucosyl and internal fructosyl units of l-kestose were asymmetric (Fig. 5 A and B), but were nearly equal throughout the labeling period in both sucrose and l-kestose in vacuoles given sucrose (Fig. 5C). The terminal fructosyl of sucrose and internal fructosyl units of l-kestose exhibited progressive loss of specific activity relative to their respective glucosyl unit, indicating that these fructosyl units may also be derived extensively from unlabeled endogenous pools of sucrose or from hydrolysis of inulin oligomers. This metabolism was apparently arrested in vacuoles provided sucrose in the absence of necessary cytoplasmic enzymes (Fig. 5C). Taken together, the results of these experiments indicate that fructan metabolism is preserved in isolated vacuoles, but just how radioactivity is incorporated into l-kestose is not clear. Without information on the specific activities of the individual sugar residues, one may conclude that SST is still active in this in vitro system. The failure to incorporate substantial amounts of radioactivity in the terminal fructosyl unit of the l-kestose in any of the systems raises doubt that SST alone is providing the l-kestose after absorption of La· beled sucrose into the vacuole. Edelman and Jefford (1966 b) cautioned that l-kestose can be made by FFT merely by transfer of the terminal fructosyl unit of l-kestose to sucrose, and Pont is (1970) came to similar conclusions through comparison of the specific activities of fructose and glucose moieties of sucrose and l-kestose in Jerusalem artichoke tuber explants given 14C-o-glucose for up to 8 days. Such reactions are futile because there is no net gain in oligomer size. Detection of SST activity can be made only by indirect determina-

I·keslose

~• •

I·keslose

sucrose

8-

f-

sucrose

....

·0

I

0

~o

0

-8--·

209

10

20

JO 0

I

10

20

Incubalion Time, min

tion of glucose in isolated vacuoles or by an increment in total l-kestose. In these short incubations, vacuoles yielded only trace amounts of labeled glucose or fructose (Fig. 1 C). The pathway of synthesis of inulin has been envisioned as SST and FFT working in series, SST providing the l-kestose and FFT using l-kestose for chain extension. Our data indicate that these enzymes may also work in parallel in vivo and in vitro, but for reasons that are not clear. The relative loss of radioactivity in the fructosyl moieties of sucrose in the tissue slices and protoplasts is an additional enigma. A simple explanation is that increased fructan hydrolase activity in the vacuole may produce large pools of unlabeled fructose in the cytoplasm of the intact cells and protoplasts that reduces incorporation of radioactive fructose into sucrose by isotope dilution. Although levels of sucrose and l-kestose remained nearly constant at 20 mM and 10 mM, respectively, during incubation of the tissues slices, the concentration of fructose rose from about 9 mM initially to 17 mM after 4 h incubation. Labeled glucose would be incorporated directly into UDP-Glc via hexokinase, phosphoglucomutase, and UDP-Glc pyrophosphorylase. Fru-6-P could be made by Glc-6-P isomerase, but a large pool of unlabeled fructose from hydrolysis of inulin would enter the cytoplasm and be incorporated via a hexokinase into Fru-6-P. Hence, sucrose-P, and ultimately sucrose, made from heavily labeled UDP- 14C-Glc and relatively unlabeled Fru-6-P would form asymmetrically labeled sucrose and l-kestose (Fig. 5 A and B). Pontis (1970) did not observe asymmetrically labeled sucrose in his studies with explants fed 14C-o-glucose although the average specific activities of the fructosyl units of l-kestose were initially about half that of glucose, reasonably consistant with our data and those of Dickerson and Edelman (1966). The nutrient medium of Pontis (1970) may have blocked hydrolysis of nascent inulin so that most of the Fru-6-P came from exogenous labeled glucose. Our success in preserving fructan metabolism in isolated vacuoles does, however, now give us a system to examine more closely the role of the tonoplast in vectorial transport of sucrose into vacuoles, glucose and

210

NICHOLAS C. CARPITA, FELIX KELLER, DAVID M. GIBEAUT, THOMAS L. HOUSLEY, and PHILIPPE MATILE

fructose out of vacuoles, and the association of these transport processes with formation of 1-kestose and fructans by SSTandFFT. Acknowledgements This work was supported by Grants 87-CRCR-1-2438 and 8937130-4749 from the United States Department of Agriculture/ Competitive Research Grants Organization and Grant INT8900004 from the National Science Foundation U.S.-Switzerland Cooperative Research Program. Journal paper No. 12,515 of the Purdue University Agriculture Experiment Station. We thank Helen Greutert, University of Zi.irich, and Anna Olek, Purdue University, for their technical assistance, and Dr. Jan Kanabus for helpful suggestions and advice.

References CARPITA, N. c., J. KANABUS, and T. L. HOUSLEY: Linkage structure of fructans and fructan oligomers from Triticum aestivum and Festuca arundinacea leaves. J. Plant Physiol. 134, 162-168 (1989). CARPITA, N. C. and E. M. SHEA: Linkage structure of carbohydrates by gas chromatography-mass spectrometry (GC-MS) of partially methylated alditol acetates. In: BIERMAN, C. J. and G. D. MCGINNIS (eds.), Analysis of Carbohydrates by GLC and MS, CRC Press, pp. 157 -216, Boca Raton, Florida (1989). DARWEN, C. W. E. and P. JOHN: Localization of the enzymes of fructan metabolism in vacuoles isolated by a mechanical method from tubers of Jerusalem artichoke. Plant Physiol. 89, 658-663 (1989). DICKERSON, A. G. and J. EDELMAN: The metabolism of fructose polymers in plants. VI. Transfructosylation in living tissue of He· {iantbus tuberosus L. J. Exptl. Bot. 17, 612 - 619 (1966). EDELMAN, J. and A. G. DICKERSON: The metabolism of fructose polymers in plants. Transfructosylation in tubers of Heliantbus tuberosus L. Biochem. J. 98, 787 -794 (1966). EDELMAN, J. and T. G. JEFFORD: The mechanism of fructosan metabolism in higher plants as exemplified in Heliantbus tuberosus L. New Phytol. 67, 517 -531 (1968). FREHNER, M., F. KELLER, and A. WIEMKEN: Localization of fructan metabolism in the vacuoles isolated from protoplasts of Jerusalem artichoke tubers (Heliantbus tuberosus L.). J. Plant Physiol. 116, 197-208 (1984).

GORDON, A. J.: Diurnal patterns of photosynthate allocation and partitioning among sinks. In: CHRONSHAW, J., W. J. LUCAS and R. T. GIAQUINTA (eds.), Phloem Transport, pp. 499-517, Alan R. Liss, New York (1986). HENDRIX, J. E., J. C. LINDEN, D. H. SMITH, C. W. Ross, and I. K. PARK: Relationship of preanthesis fructan metabolism to grain number in winter wheat (Triticum aestivum L.). Aust. J. Plant Physiol. 13, 391-398 (1986). HENDRY, G.: The ecological significance of fructan in a contemporary flora. New PhytoL, Suppl. 106, 201-216 (1987). JERMYN, M. A.: A new method for the determination of ketohexoses in the presence of aldohexoses. Nature 177, 38-39 (1956). KANABUS, J., D. M. GIBEAUT, N. C. CARPITA, and T. L. HOUSLEY: Fructosyl transfer between 1-kestose and sucrose in wheat leaves. Plant Physiol., in press. KVERNHIEM, A. L.: Methylation analysis of polysaccharide with butyllithium in dimethyl sulfoxide. Acta Chern. Scand., Ser. B 41, 150-152 (1987). LEWIS, D. H.: Occurrence and distribution of carbohydrates in vascular plants. In: LEWIS, D. H. (ed.), Storage Carbohydrates in Vascular Plants, Soc. Expl. BioI. Sem. Series 19, pp. 1-52, Cambridge Press, Cambridge (1984). MEIER, H. and J. S. G. REID: Reserve polysaccharides other than starch in higher plants. In: LOEwus, F. A. and W. TANNER (eds.), Encyclopedia of Plant Physiology, New Series, Vol. 13 A, pp. 418-471, Springer-Verlag, Berlin (1982). POLLOCK, C. A. and N. J. CHATTERTON: Fructans. In: PREISS, J. (ed.), The Biochemistry of Plants, pp. 109-140, Academic Press, New York (1988). PONTIS, H. G.: The role of sucrose and fructosylsucrose in fructosan metabolism. Physiol. Plant. 23, 1089-1100 (1970). SHEA, E. M., D. M. GIBEAUT, and N. C. CARPITA: Structural analysis of the cell walls regenerated by carrot protoplasts. Planta 179, 293 -308 (1989). SHIOMI, N.: Properties of fructosyltransferases involved in the synthesis of fructan in Liliaceous plants. J. Plant Physiol. 134, 151-155 (1989). WAGNER, W., F. KELLER, and A. WIEMKEN: Fructan metabolism in cereals: induction in leaves and compartmentation in protoplasts and vacuoles. Z. Pflanzenphysiol. 112, 359-372 (1983). WIEMKEN, A., M. FREHNER, F. KELLER, and W. WAGNER: Fructan metabolism, enzymology and compartmentation. In: Current Topics in Plant Biochemistry and Physiology, Vol. 5, pp. 17 - 37, University of Missouri, Columbia, MO (1986).