Efficient uptake of flavonoids into parsley (Petroselinum hortense) vacuoles requires acylated glycosides

Planta 9 Springer-Verlag1986

Planta (1986) 167:183M89

Efficient uptake of flavonoids into parsley (Petroselinum hortense) vacuoles requires acylated glycosides U. Matern, C. Reichenbach and W. Heller Department of Plant Biochemistry, Biological Institute II, University of Freiburg, Schfinzlestrasse 1, D-7800 Freiburg, Federal Republic of Germany

Abstract. Vacuoles were prepared from cultured parsley cells by polyamine-induced rupture of protoplasts. Acid-phosphatase activity, associated exclusively with the vacuoles, served for determination of vacuole yield in subsequent transport studies. Isolated vacuoles rapidly accumulated [2"-14C]apigenin 7-O-(6-O-malonylglucoside) or [2"-14C]fl-methyl D-6-O-malonylglucoside added at approximately 20 nM and 1.5 gM concentration, respectively, to the incubation mixture. The accumulation was linear with time and strongly dependent on alkaline buffer conditions as well as on the age of the vacuole preparation. Subsequent addition of a malonic hemiester esterase did not relase the label from the vacuoles. Moreover, neither [2-14C]apigenin 7-O-glucoside or [2-14C]ma lonic acid accumulated in the vacuoles under any assay conditions, nor did such compounds or fimethyl D-glucopyranoside, a malonic diester, and a succinic monoester inhibit transport of the acylated flavonoid. Transport was, however, inhibited by /?-methyl D-6-O-malonylglucopyranoside. Vacuoles which had been incubated for more than 40 rain at pH 8.0 did not stain any more with neutral-red dye and concomitantly lost the previously accumulated acylated glucoside. Our data confirm that malonylglucoside uptake by parsley vacuoles involves selective tranpsort sites. It is suggested that changes in the molecular symmetry of the malonylglucosides are responsible for vacuolar trapping of flavonoids in parsley. Key words: Flavonoid 7-O-(6-O-malonylglycosides) - P e t r o s e l i n u m - Tonoplast (acylated glucosides). Introduction A variety of compounds have been identified from plant vacuoles, which have been of help in assignAbbreviation: DEAE = diethylaminoethyl

ing a physiological role to this organelle. Vacuoles have been considered, for example, as the lytic compartment (Matile 1975), the long-term storage compartment for assimilates (Keller and Wiemken 1982; Frehner et al. 1984), and also as a short-term storage buffer for compounds synthesized and metabolized at different times of the day and night (Kenyon et al. 1978; Holl/inder-Czytko and Amrhein 1983). It has further been suggested that vacuoles constitute a "toxic compartment" (Matile 1984), serving the plant's need to isolate temporarily those substances that do not support metabolism or even inhibit it, such as alkaloids (Saunders 1979; Neumann et al. 1983), and glycosidic steroid derivatives (L6ffelhardt et al. 1979; Kesselmeier and Urban 1983), hormone derivatives (GarciaMartinez et al. 1981) and phenolics (Hrazdina et al. 1982; Schmitt and Sandermann 1982). On the assumption that most of these compounds are synthesized in the cytoplasm, their efficient and selective transport through the tonoplast must be postulated. A diffusion model for tonoplast transport, relying solely on the pH gradient between cytoplasm and vacuole, has been inferred from the intracellular distribution of synthetic basic dyes (Sitte 1972). While initially it was suggested that this model could apply generally to the tonoplast transport of various amines (Kurkdijan and Guern 1981), more recent publications point to an inward uniport through specific carriers of ammonia, methylamine (Kleiner 1981), and also alkaloids (DeusNeumann and Zenk 1984). Unfortunately, in these examples neither the driving force nor the molecular basis of selectivity has so far been defined. A different model must apply to vacuolar uptake and trapping of organic acids, a process which is accomplished against the proton concentration gradient, as exemplified by malate accumulation in Crassulacean acid metabolism (CAM) plants (Lfittge et al. 1981). This transport requires energy

184 and appears to be mediated by a specific malic-acid permease (Buser-Suter et al. 1982). At present no model has been put forward for tonoplast transport of glycosides, and transport was demonstrated in only a few instances employing low ligand concentrations (Werner and Matile 1985). At non-physiologically high concentrations, passive tonoplast transport of phenol glucosides was reported (Alibert et al. 1982). Transport under these conditions was steady over long periods of incubation, was concentration-dependent and could not be saturated. Like many other plants, parsley accumulates glycosidic malonic hemiesters of various phenolic pigments rather than the respective glucosides (citations in Matern 1983 and KSster et al. 1984). Although synthesized in the cytoplasm, these hemiesters are stored exclusively in the vacuoles (Matern et al. 1983a). Recently, we demonstrated that in vitro apigenin 7-O-(6-O-malonylglucoside), the main pigment in parsley, undergoes a change in molecular conformation dependent on the proton concentration of its microenvironment. We proposed, therefore, that in vivo stereoselective tonoplast passage of only one of the flavonoid conformers in concert with a conformational transition induced by vacuolar pH is responsible for accumulation of flavonoids in this organelle. Selective vacuolar accumulation of only the cis-isomer of a coumaric acid glucoside has recently been reported in Melilotus (Alibert et al. 1985). This report extends our investigations on the physiological function of malonic acid conjugates by homologous transport studies employing isolated parsley vacuoles.

Material and methods Chemicals. Dextran sulfate 500, sodium salt, 4-nitrophenyl phosphate, 2-(N-morpholino)ethanesulfonic acid (Mes), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes), fl-methyl Dglucopyranoside and Celite type 545 were purehasesd from Serva, Heidelberg, FRG. Ficoll 400 and diethylaminoethyl (DEAE)-Dextran were from Pharmacia, Freiburg, FRG, and ATP, fructose-6-phosphate and malonyl-CoA came from Sigma, Miinchen, FRG. Malonic acid, apigenin 7-O-glucoside and pectinase were purchased from Roth, Karlsrhe, FRG, and driselase, as well as dicyclohexyl carbodiimide and succinic anhydride, were from Fluka, Neu-Ulm, FRG. Pectinase and driselase were extensively dialyzed against water prior to use. Mannitol, sorbitol and propionic acid were purchased from Merck, Darmstadt, FRG. [2-14C]Malonic acid (2.07TBqmol- 1) and [2-14C]malonyl-CoA (2.15 TBq. mol- ~)were purchased from Amersham-Buehler, Braunschweig, FRG. [214C]Apigenin 7-O-glucoside (74 GBq.mo1-1 and [2"'-14C]apigenin 7-O-(6-O-malonylglucoside) (74 GBq.mol 1 and

U. Matern et al. : Tonoplast transport of acylatedglucosides 2.15 TBq-mo1-1) were prepared enzymatically, and the acylated flavonoid was purified by electrophoresis as described previously (Matern et al. 1983a) prior to use. fl-Methyl D-6-Omalonylglucoside was synthesized according to Kasai et al. (1981) and purified subsequently by paper chromatography.

[2"-14C]fl-Methyl D-6-O-malonylglucoside. [2-14C]Malonic acid (0.224 retool/74 MBq) and fl-methyl D-glucopyranoside (0.26 retool) were dissolved in dry dioxane (1 ml) and dicyclohexyl carbodiimide (0.26 retool) was added. After stirring for 2 d at room temperature, water (1 ml) was added, the suspension filtered through celite, and [2"-14C]fl-methyl D-6-O-malonylglucoside was purified from the filtrate by repeated chromatography on Dowex 1 x 2 as described by Kasai et al. (1981). Cochromatography with authentic material (Matern et al. 1983 a) and subsequent autoradiography confirmed its identity. Since this material still contained a small amount of an impurity which strongly interfered with uptake, the acylated glncoside was further purified by chromatography on Whatman 3 MM paper in n-butanol:acetic acid: water, 4:1 : 5 (by vol., upper phase), eluted from the paper (section Rf 0.1 0.25) with water: acetic acid, 99 : 1, and lyophilyzed shortly before use. Malonic acid monobenzyl ester. This ester was synthesized as described previously (Matern et al. 1984). Malonic acid dibenzyl ester. Malonic acid (5 mmol), benzyl alcohol (18.5 mmol), trichloromethane (5 ml) and SOC12 (0.5 ml) were mixed and stirred overnight at room temperature. Ethyl acetate (95 ml) was added, and the organic phase was washed successively with water (3 times 100 ml) and 10% potassium bicarbonate (3 times 100 ml). The dibenzyl ester was purified from the organic phase by chromatography on a silica-gel column (15 g) by a step gradient made up with diethyl ether and petroleum ether (50-60 ~ C). Succinic acid monobenzyl ester. Succinic acid anhydride (1 g) dissolved in dry pyridine (2 ml) and benzyl alcohol (1 ml) was stirred for 24 h at room temperature and then water (30 ml) and ethyl acetate (50 ml) were added. The organic phase was washed subsequently with 2 N HC1 (10 ml), the monobenzyl ester was extracted with 10% potassium bicarbonate (5 times 10 ml) and reextracted from the acidified aqueous phase with ethyl acetate (50 ml). The organic phase was washed with water (3 times 10 ml), dried over sodium sulfate and evaporated. The monobenzyl ester was purified from the residue by recrystallization in ethyl acetate: petroleum ether (50-60 ~ C), and its identity was confirmed by UV and IR spectroscopy. Buffers. The following buffers were used : (A) 50 mM potassium phosphate pH 5.7, containing 1 mM citrate, 1 mM MgC12, 0.35 M sorbitol and 0.35 M mannitol; (B) 25 mM Hepes-2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris) pH 7.0, containing 0.7 M mannitol; (C) 25 mM Hepes-Tris pH 8.0, containing 0.7 M mannitol; (D) 25 mM Mes-Tris pH 6.5, containing 0.7 M mannitol; (E) 50 mM triethanolamine HCl-sodium hydroxide pH 7.6; (F) 20raM Hepes-Tris pH 8.0, containing 1.5mM dithiothreitol (DTT); (G) 100 mM succinie acid-sodium hydroxide pH 5.0; (H) 20 mM citrate-phosphate (McIlvain) buffer pH 5.0. Isolation of vacuoles. Protoplasts were prepared from 6 d old dark-grown parsley (Petroselinum hortense) cell cultures (2-3 g cell fresh weight for each individual experiment) with pectinase (30 rag) and driselase (90 mg) in buffer A (6 ml) (Matern et al. 1983a). Protoplasts were recovered from the resulting suspension by filtration through a 70-lain nylon net and subsequent

U. Matern et al. : Tonoplast transport of acylated glucosides centrifugation (approx. 5,500 g for 20 rain) onto a Ficoll cushion (26% in 3 ml buffer A). Protoplasts were resuspended several times in buffer A (5 ml) and sedimented (approx. 2,000g for 10 min) at room temperature. Finally, the protoplasts were suspended in buffer B (2 ml), and the vacuoles were prepared by centrifugation through a DEAE-Dextran/Dextran sulfate gradient similar to the procedure described originally by Boudet et al. (1981). The gradient was made up from Ficoll steps in buffer B of 12.5% (2 ml), 6.7% (2 ml), 5.4% containing 4 mg. m1-1 Dextran sulfate (2 ml) and 4.8% containing 4 mg-m1-1 DEAE-Dextran (5 ml). Centrifugation was carried out at room temperature (2,000 g for 30 min). Vacuoles were carefully recovered from the 12.5%/6.7% Ficoll interface and were used immediately for the uptake experiments. From aliquots of the vacuole suspension, acid phosphatase was routinely determined in buffer G after lysis of the vacuoles. Numbers of vacuoles as well as of protoplasts were evaluated in a Fuchs-Rosenthal hemocytometer (0.2 mm deep) when necessary.

Enzyme determinations. Protoplasts and vacuoles were broken either in an Elvehjem potter or by freezing and thawing in buffer F. After centrifugation, the following enzyme activities were determined from aliquots of the supernatant using the procedures described by Boller and Kende (1979): c~-galactosidase, fl-galactosidase, fl-glucosidase, c~-mannosidase,/]-mannosidase, fl-acetylglucosaminidase and acid phosphatase. Phosphoglucose isomerase was determined in buffer E according to Bergmeyer (1974).

Photomicroscopy. Photography of vacuoles was carried out with an inverted microscope Zeiss IM 35 with DIK equipment (Zeiss, Oberkochem, FRG) according to Nomarski.

Standard assay for tonoplast tramport. For determination of tonoplast transport, 1 ml of the freshly prepared vacuole suspensions (representing usually between 105 and 10 6 vacuoles) was added to 4 ml of buffer C containing 1 mM ATP, I mM MgC12 and between 20 and 600 nM concentration of either one of the ligands [2-1~C]apigenin 7-O-glucoside, [2"'-14C]api genin 7-O-(6-O-malonylglucoside) and [2-14C]malonic acid, or 1.5 btM [2"-~4C]/?-methyl D-6-O-malonylglucoside. The preparation was shaken gently on a rotary shaker at room temperature and the incubation terminated after 20 rain by layering the mixture onto the top of a Fieoll gradient in buffer B (2 ml each of 12.5%, 6.7%, and 3 ml each of 5.4% and 4.8%) and subsequent centrifugation at 2,000g for 20 rain. Between 30 to 40% of the vacuoles initially added to the incubation mixture were usually recovered from the 12.5%/6.7% Ficoll interface of the gradient. Phosphatase activity and relative distribution of radioactivity were determined after fractionation. Determination of radioactivity. Radioctivity of solutions and vacuole suspensions was determined from aliquots by liquid scintillation counting in dioxane containing 5 g of 2,5-diphenyloxazole. 1-1 and 100 g of naphthalene. 1-1.

Acylhydrolase. Leaves and stems of five-month-old parsley plants (1 g) were ground with quartz sand in buffer H (3 ml). After centrifugation, esterase activity was determined as previously described (Matern 1983) but adding aliquots of the supernatant to the tonoplast-transport assay mixture and omitting addition of the vacuoles.

Inhibition of tonoplast transport. Substances to be tested for transport inhibition were added in 50 gl water to the transport assay mixture containing 20 nM of [2"'-14C]apigenin 7-0-(6-0-

185 malonylglucoside). The solutions were carefully neutralized with potassium hydroxide prior to addition. Due to its poor solubility in water, malonic acid dibenzyl ester was dissolved in ethyleneglycol monomethyl ether (30 bd). This amount of solvent did not interfere with transport under the conditions employed.

Results

Isolation and characterization of vacuoles.Several procedures for vacuole preparation from protoplasts were examined for their efficiency in isolating vacuoles from parsley cells. These included osmotic rupture (Saunders 1979), vacuum filtration through glass wool (Moskowitz and Hrazdina 1981), squeezing through a syringe (Kaiser and Heber 1984), and polybase-induced lysis (Dfirr et al. 1975). Only the centrifugation of protoplasts through a DEAE-Dextran/Dextran sulfate step gradient similar to the procedure described by Boudet et al. (1981) was found to yield vacuole suspensions of sufficient density (10 6 vacuoles, ml- 1 on the average). Moreover, the speed of this procedure was essential for the outcome of subsequent uptake studies. From approx. 6.10 6 protoplasts, up to 2.10 6 vacuoles were recovered from the first centrifugation employing buffer B. Buffer changes with respect to either pH (between pH 6 and pH 8) or addition of calcium, magnesium, ATP, ethylenediaminetetraacetic acid (EDTA) or glucose did not significantly increase vacuole yield. Vacuoles were usually sedimented a second time through a Ficoll gradient lacking DEAE-Dextran and Dextran sulfate with a recovery of about 50%. Such vacuoles were free of cytoplasmic components, as determined by monitoring phosphoglucose isomerase activity. Freshly isolated vacuoles kept in buffer B could be efficiently stained with neutral-red dye, but the dye was rapidly released again after more than about 30 rain following the isolation. Moreover, on examination through differential interference contrast optics, less-dense areas were distinguished within the freshly purified vacuoles (Fig. 1), pointing to either the presence of less-dense materials adhering to the tonoplast or vacuolar inclusions of reduced density. On the basis of our previous photographs (Milder 1984), the latter explanation appears more likely. Search for marker enzymes. With reference to a previous report on the vacuolar localization of lytic enzymes (Boller and Kende 1979), parsley protoplasts were examined for enzyme activities such as 0~-galactosidase, /?-galactosidase, c~-mannosi-

186

U. Matern et al. : Tonoplast transport of acylated glucosides

Fig. 1. Freshly isolated parsley vacuoles containing inclusions of reduced optical density. Photomicroscopy was carried out by P. Sitte (Department of Cell Biology, University of Freiburg) using a Zeiss photomicroscope with Nomarski optics. Bar = 10 gm

HO2CN2'"

HO2C\2.

/CH2

/CH2 O=C N 6' /0 CH2

O=C,, 6" /O CH2 "

HO~ ''~

q

.~~4'~OH

8

HO-~oHr' O ~

A

l

H

OH

0

r"

,Y

H 0 ~'-~-~- 0

HO--~O\cH3 OH

B

dase, fl-mannosidase, #-glucosidase, #-acetylglucosaminidase, and acid phosphatase, following lysis of the protoplasts in buffer F. Appreciable activity of only the last enzyme could be measured (on average 3 nkat. m g - 1 protein). After fractionation, more than 95 % of the total acid phosphatase activity of protoplasts could repeatedly be ascribed to the vacuoles. As a result of the difficulties encountered in accurate determination of vacuole yield, even more than 100% of this activity was calculated for the vacuole fraction in several experiments. On the other hand, the cytoplasmic localization of phosphoglucose isomerase, an enzyme functional in primary metabolism, was confirmed for parsley protoplasts (approx. 4 n k a t ' m g - ~ protein). This enzyme activity, as well as the acid phosphatase activity, routinely served for control of clean separation of cytoplasmic components and vacuoles.

Transport of flavonoids. [2-14C]Apigenin 7-O-glucoside and [2"-14C]apigenin 7-O-(6-O-malonyl-

Fig. 2. Malonic acid hemiesters which were accumulated by isolated parsley vacuoles. A apigenin 7-O-(D-6-O-malonylglucoside) and B #-methyl D-6-O-malonylglucoside

glucoside) (Fig. 2) were dissolved in water:acetic acid, 100: 2 (v: v) following their chromatographic or electrophoretic purification, and were extracted from the aqueous phase into n-butanol shortly before use. [2-14C]Apigenin 7-O-glucoside was not taken up by isolated vacuoles under any assay condition or at any concentration used (up to 200 nM). [2""t4C]Apigenin 7-O-(6-O-malonylglucoside), on the other hand, added at only 20 nM or up to about 200 nM concentration was rapidly accumulated by the vacuoles. The apparent uptake was linear with time for up to 20 rain (Fig. 3) and was dependent on vacuole numbers, while the addition of ATP and MgC12 did not change its rate. Moreover, maximal rate of accumulation of the malonylated flavonoid was observed at pH 8.0, and declined steeply when the pH of the incubation mixture was adjusted to a value below pH7.5 (Fig. 4). Although at pH 6.5 the vacuole vesicles appeared to be more stable, the accumulation rate of the malonylated flavonoid was negligible under these conditions (Fig. 4).

U. Matern et al. : Tonoplast transport of acylated glucosides

o~ 0)

\

2-

0

0

5

10

15

20

Incubotion Time [ rain ] Fig. 3. Time-dependent uptake of [2"'-14C]apigenin 7-0-(6-0malonylglucoside) (2.15 TBq-mol-1) by freshly isolated parsley vacuoles. The acylated flavonoid had been added to a final concentration of approx. 20 nM in the transport assay mixture (5 ml) at pH 8.0. Aliquots of one vacuole preparation were used for each set of determinations

_~ 80 I)

>06x

'6

~26;s

7.'5

,#

oo

pH of Incubation Buffer Fig. 4. pH-Dependent uptake of [2"-14C]apigenin 7-0-(6-0-

malonylglucoside) (2.15 TBq. mol- 1) by freshly isolated parsley vacuoles. The acylated flavonoid had been added to a final concentration of approx. 20 nM in the assay buffer, and the assays were incubated for 20 rain under standard conditions. Aliquots of one vacuole preparation were used for each set of determinations Freshly isolated vacuoles kept at pH 8.0 could be used for uptake studies for up to about 25 min. On incubation exceeding this time span, the label accumulated was again released from the vacuoles. Addition of various concentrations of ATP and MgC12, or the addition of glucose, citrate, and EDTA, respectively, did not influence these results appreciably.

FIavonoid transport in the presence of malonic acid andparsley acylhydrolase. The initial transport assays did not exclude the possibility that the malonylated flavonoid adhered to the tonoplast membranes rather than being taken up into the vacuoles. Moreover, it remained to be confirmed that no hydrolysis of the malonic hemiester prior to vacuolar accumulation of the label occurred under the assay conditions, although no such esterase ac-

187

tivity could previously be detected (Matern 1983) in cultured parsley cells. [2-14C]Malonic acid (222GBq-mo1-1) provided at concentrations ranging from roughly 180 to 600 nM was not accumulated by the isolated vacuoles. In separate sets of experiments, vacuoles were incubated with 20 nM of [2"-14C]apigenin 7-O-(6-O-malonylglucoside) for 15 min at which time a crude malonic hemiester esterase (Matern 1983) was added and incubation continued for an additional 5 rain. Control assays were terminated after the initial 15 rain of incubation. Up to 500 gl of buffer H containing the esterase did not change the pH of the tranpsort assay mixture. The amount of esterase added (approx. 37 nkat) was sufficient to hydrolyze the entire malonic acid conjugate in the assay within 5 min. Addition of the esterase did not decrease the amount of radioactivity accumulated by the vacuoles as compared with the controls. Furthermore, when such experiments were carried out in the presence of excess non-labelled malonic acid (500 gM) or apigenin 7-O-glucoside (50 gM, a concentration close to the limits of its solubility), no appreciable differences were observed. Conceivably, the malonylated flavonoid present in these assays at only 20 nM concentration was taken up into the vacuoles independently of the mechanism which in general may apply to uptake of phenol glycosides at elevated concentration.

Transport of malonylated fl-methyl D-glucopyranoside. Vacuolar uptake of [2"-14C]fl-methyl D-6-Omalonylglucoside (1.5 ~tM, 0.33 TBq. m o l - ~; Fig. 2), was also linear over about 20 rain, and showed a pronounced p H dependence with maximal rates between p H 7.5 and p H 8.0. Non-labelled fl-methyl D-glucopyranoside (up to 5 m M added) did not diminish its rate of uptake. Similar to the results obtained with the malonylated flavonoid, neither the addition of parsley esterase nor its combination with non-labelled malonic acid changed these results appreciably.

Inhibition of tonoplast transport. While the addition of either propionic acid (up to 500 ~tM), malonic acid (up to 5 mM), succinic acid monobenzyl ester (up to 5 raM) and fl-methyl D-glucopyranoside (up to 5 raM), respectively, had no effect on tonoplast transport of the malonylated flavonoid, transport was slightly inhibited by large concentrations (exceeding 5 mM) of malonic acid dibenzyl ester, and was moderately inhibited by malonic acid monobenzyl ester at concentrations exceeding 500 ~tM. The uptake was, however, completely inhibited in

188

the presence of 2 mM/?-methyl D-6-O-malonylglucoside. Discussion

The isolation of carbohydrate malonic acid hemiesters from plants has been reported frequently (citations in Matern 1983; K6ster et al. 1984; W61decke and Herrmann 1974; Tamura etal. 1983; Bridle et al. 1984; Ghisla et al. 1984). The physiological significance of such conjugates, however, has so far remained obscure. Recently, we proposed that malonic acid conjugation in parsley mediates tonoplast transport of flavonoid glycosides (Matern et al. 1983 b). The present report confirms our previous proposal by uptake studies employing vacuoles isolated from cultured parsley cells. The observation that vacuoles were functional in transport for only a short period of time following their isolation is not surprising. For example, soybean vacuoles (Schmitt and Sandermann 1982) maintain their acidic pH for only a short while (Schmitt, R., Institut ffir Biochemische Pflanzenpathologie der GSF (Gesellschaft fiir Strahlen- und Umweltforschung Miinchen), Neuherberg, FRG, personal communication), and exogeneously supplied ATP and MgC12 did not reverse or delay this effect in either soybean or parsley vacuoles. The apparent vacuolar accumulation of apigenin 7-O-(6-O-malonylglucoside) was not hindered by the presence of either apigenin 7-O-glucoside or malonic acid in large excess, or by subsequent addition of an appropriate acylhydrolase. These results clearly indicate that the malonylated pigment was being taken up rather than simply being attached to the tonoplast surface. The observed tonoplast transport was very dependent on the pH of the incubation buffer. Efficient transport was found only at alkaline pH, a finding which contrasts sharply with previous reports on tonoplast transport of sugars (Thorn et al. 1982; Guy et al. 1979; Doll et al. 1979). Moreover, flavonoid transport was independent of exogeneously supplied ATP and magnesium. Whereas for sugar transport, protonation of particular transport components was considered necessary (Reinhold and Kaplan 1984), we suggest that the apparent pH dependence of flavonoid transport in parsley primarily reflects the need for the flavonoid conformation that is induced by alkaline conditions. Molecular-symmetry changes of apigenin 7-O-(6-O-malonylglucoside) have been reported previously (Matern et al. 1983a) in response to changes in the proton concentration of its microenvironment. Acid conditions in the vacuole may be

U. Matern et al. : Tonoplast transport of acylated glucosides

responsible for subsequent vacuolar trapping of the malonylated flavonoids by favouring a switch in their conformation. This proposal is corroborated by the observation that during extended incubation in alkaline buffer, isolated vacuoles, although visibly unchanged, lost their acidic pH concomitantly with their flavonoid content. It is of interest to note in this context that optimal uptake of esculin into barley vacuoles (Werner and Matile 1985), and the uptake of a gibberellic acid into barley and cowpea vacuoles (Knuth et al. 1983) was reported at alkaline pH. Vacuolar uptake of flavonoids in parsley probably proceeds via specific tonoplast transport sites and seems to depend primarily on the malonylglucose moiety, since/?-methyl D-6-O-malonylglucoside also accumulated in the vacuoles. Neither malonic acid, malonic acid dibenzyl ester nor succinic acid monobenzyl ester inhibited uptake of the malonylglucosides. Affinity to the transport sites may be strongly influenced, however, by the alcohol portion of the hemiester, since malonic acid monobenzyl ester required comparatively large concentrations for inhibition of malonylglucoside uptake. Saturation kinetics in homologous and even in a non-homologous system (Werner and Matile 1985) have been used to argue in favour of specific tonoplast transport. Phenol glucosides, however, may show non-saturable kinetics when employed at high concentration (Alibert et al. 1982). Instead we demonstrate in this report that/?-methyl D-6-Omalonylglucoside efficiently inhibited transport of the malonylated flavonoid glucoside. These results strongly indicate that tonoplast transport of malonylglucosides in parsley is mediated by a specific system of transport sites. This work was supported by the Wissenschaftliche Gesellschaft Freiburg and by the Fonds der Chemischen Industrie. We thank C. Beggs for critically reading the manuscript. We are indebted to P. Sitte, Department of Cell Biology, Freiburg, for photomicroscopy of vacuoles.

References Alibert, G., Boudet, A.M., Canut, H., Rataboul, P. (1985) Protoplasts in studies of vacuolar storage compounds. In: The physiological properties of plant protoplasts, pp. 105-115 Pilet, P.E., ed. Springer, Berlin Heidelberg New York Tokyo Alibert, G., Boudet, A.M., Rataboul, P. (1982) Transport of o-coumaric acid glucoside in isolated vacuoles of sweet clover. In: Plasmalemma and tonoplast transport: their functions in the plant cell. Developments in plant biology, vol. 7, pp. 193-200, Marm6, D., Marrb, E., Hertel, R., eds. Elsevier, Amsterdam New York Oxford Bergmeyer, H.U. (1974) Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim Boller, T., Kende, H. (1979) Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol. 63, 1123-1132

U. Matern et al. : Tonoplast transport of acylated glucosides

g0udet, A.M., Canut, H., Alibert, G. (1981)Isolation and characterization of vacuoles from Melilotus alba mesophyll. Plant Physiol. 68, 1354-1358 Bridle, P., Loeffler, R.S.T., Timberlake, C.F., Self, R. (1984) Cyanidin 3-malonylglucoside in Cichorium intybus. Phytochemistry 23, 2968-2969 Buser-Suter, C., Wiemken, A., Matile, Ph. (1982) A malic acid permease in isolated vacuoles of a crassulacean acid metabolism plant. Plant Physiol. 69, 456-459 Deus-Neumann, B., Zenk, M.H. (1984) A highly selective alkaloid uptake system in vacuoles of higher plants. Planta 162, 250-260 Doll, S., Rodier, F., Willenbrink, J. (1979) Accumulation of sucrose in vacuoles isolated from red beet tissue. Planta 144, 407-411 Diirr, M., Boller, T., Wiemken, A. (1975) Polybase induced lysis of yeast sphaeroplasts. Arch. Microbiol. 105, 319-327 Frehner, M., Keller, F., Wiemken, A. (1984) Localization of fructan metabolism in the vacuoles isolated from protoplasts of Jerusalem artichoke tissue. J. Plant Physiol. 116, 197-208 Garcia-Martinez, J.L., Ohlrogge, J.B., Rappaport, L. (1981) Differential compartmentation of gibberellin A1, and its metabolites in vacuoles of cowpea and barley leaves. Plant Physiol. 68, 865 867 Ghisla, S., Mack, R., Blankenhorn, G., Hemmerich P., Krienitz, E., Kuster, T. (1984) Structure of a novel flavin chromophore from Arena coleoptiles, the possible "blue light" photoreceptor. Eur. J. Biochem. 138, 339-344 Guy, M., Reinhold, L., Michaeli, D. (1979) Direct evidence for a sugar transport mechanism in isolated vacuoles. Plant Physiol. 64, 61-64 Holl/inder-Czytko, H., Amrhein, N. (1983) Subcellular compartmentation of shikimic acid and phenylalanine in buckwheat cell suspension cultures grown in the presence of shkikmate pathway inhibitors. Plant Sci. Lett. 29, 89-96 Hrazdina, G., Marx, G.A., Hoch, H.C. (1982) Distribution of secondary plant metabolites and their bisosynthetic enzymes in pea (Pisum sativum L.) leaves. Plant Physiol. 70, 745-748 Kaiser, G., Heber, U. (1984) Sucrose transport into vacuoles isolated from barley mesophyll protoplasts. Planta 161, 562-568 Kasai, T., Okuda, M., Sakamura, S., (1981) 6-O-Malonyl-flmethyl-D-glucopyranoside from roots of Rumex obtusifolius. Phytochemistry 20, 1131-1132 Keller, F., Wiemken, A. (1982) Differential compartmentation of sucrose and gentianose in the cytosoI and vacuoles of storage root protoplasts from Gentiana lutea L.. Plant Cell Rep. 1,274-277 Kenyon, W.H., Kringstad, R., Black, C.C. (1978) Diurnal changes in the malic content of vacuoles isolated from leaves of the Crassulacean acid metabolism plant, Sedum telephium. FEBS Lett. 94, 281-283 Kesselmeier, J., Urban, B. (1983) Subcellular localization of saponins in green and etiolated leaves and green protoplasts of oat (Arena sativa L.). Protoplasma 114, 133-140 Kleiner, D. (1981) The transport of NHa and NH~- across biological membranes. Biochim. Biophys. Acta 639, 41-52 Knuth, M.E., Keith, B., Clark, C., Garcia-Martinez, J.L., Rappaport, L. (1983) Stabilization and transport capacity of cowpea and barley vacuoles. Plant Cell Physiol. 24, 423432 Koester, J., Bussmann, R., Barz, W. (1984) Malonyl-coenzyme A: isoflavone 7-O-glucoside-6"-malonyltransferase from roots of chick pea (Cicer arietinum L.). Arch. Biochem. Biophys. 234, 513-521

189

Kurkdijan, A., Guern, J. (1981) Vacuolar pH measurement in higher plant cells. Plant Physiol. 67, 953-957 Loeffelhardt, W., Kopp, B., Kubelka, W. (1979) Intracellular distribution of cardiac glycosides in leaves of Convallaria majalis. Phytochemistry 18, 1289-1291 Ltittge, U., Smith, J.A.C., Marigo, G., Osmond, C.B. (1981) Energetics of malate accumulation in the vacuoles of Kalanchoe tubiflora cells. FEBS Lett. 126, 81-83 Milder, M. (1984) Die pflanzliche Vakuole aus biochemischer Sicht. Biol. Unserer Zeit 14, 171-176 Matern, U. (1983) Acylhydrolases from parsley (Petroselinum hortense). Relative distribution and properties of four esterases hydrolyzing malonic acid hemiesters of flavonoid glucosides. Arch. Biochem. Biophys. 224, 261-271 Matern, U., Feser, C., Hammer, D. (1983 b) Further characterization and regulation of malonyl-coenzyme A: fiavonoid glucoside malonyltransferases from parsley cell suspension cultures. Arch. Biochem. Biophys. 226, 206-217 Matern, U., Feser, C., Heller, W. (1984) N-Malonyltransferases from peanut. Arch. Biochem. Biophys. 235, 218-227 Matern, U., Heller, W., Himmelspach, K. (1983a) Conformational changes of apigenin 7-O-(6-O-malonylglucoside), a vacuolar pigment from parsley, with solvent composition and proton concentration. Eur. J. Biochem. 133, 439-448 Matile, Ph. (1975) The lytic compartment of plant cells. Cell biology monographs, vol. 1, Alfert, M., Beermann, W., Rudkin, G., Sandritter, W., Sitte, P., eds. Springer, Wien New York Matile, Ph. (1984) Das toxische Kompartiment der Pflanzenzelle. Naturwissenschaften 71, 18-24 Moskowitz, A.H., Hrazdina, G. (1981) Vacuolar contents of fruit subepidermal cells from Vitis species. Plant Physiol. 68, 686-692 Neumann, D., Krauss, G., Hieke, M., Gr6ger, D. (1983) Indole alkaloid formation and storage in cell suspension cultures of Catharanthus roseus. Planta Med. 48, 20-23 Reinhold, L., Kaplan, A. (1984) Membrane transport of sugars and amino acids. Annu. Rev. Plant Physiol. 35, 45-83 Saunders, J.A. (1979) Investigations of vacuoles isolated from tobacco, I. Quantitation of nicotine. Plant Physiol. 64, 74-78 Schmitt, R., Sandermann, Jr., H. (1982) Specific localization of fl-D-glucoside conjugates of 2,4-dichlorophenoxyacetic acid in soybean vacuoles. Z. Naturforsch. 37 Tell C, 772-777 Sitte, P. (1972) Vitalffirbung nach dem Ionenfallen-Prinzip. Biol. Unserer Zeit 2, 19~194 Tamura, H., Kondo, T., Kato, Y., Goto, T. (1983) Structures of a succinyl anthocyanin and a malonyl flavone, two constituents of the complex blue pigment of cornflower Centaurea cyanus. Tetrahedron Lett. 24, 5749-5752 Thorn, M., Komor, E., Maretzki, A. (1982) Vacuoles from sugarcane suspension cultures. II. Characterization of sugar uptake. Plant Physiol. 69, 1320-1325 Werner, C., Matile, Ph. (1985) Accumulation of coumaryl glucosides in vacuoles of barley mesophyll protoplasts. J. Plant Physiol. 118, 237 249 W61decke, M., Herrmann, K. (1974) Flavonole und Flavone der Gemtisearten. IV. Flavonole und Flavone des Kopfsalates und der Endivien. Z. Lebensm. Unters. Forsch. 156, 153-157

Received 30 March; accepted 30 August 1985