Different energization mechanisms drive the vacuolar uptake of a flavonoid glucoside and a herbicide glucoside.

Glycosylation of endogenous secondary plant products and abiotic substances such as herbicides increases their water solubility and enables vacuolar deposition of these potentially toxic substances. We characterized and compared the transport mechanisms of two glucosides, isovitexin, a native barley flavonoid C-glucoside and hydroxyprimisulfuron-glucoside, a herbicide glucoside, into barley vacuoles. Uptake of isovitexin is saturable (Km = 82 μM) and stimulated by MgATP 1.3-1.5-fold. ATP-dependent uptake was inhibited by bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, but not by vanadate. Transport of isovitexin is strongly inhibited after dissipation of the ΔpH or the ΔΨ across the vacuolar membrane. Uptake experiments with the heterologue flavonoid orientin and competition experiments with other phenolic compounds suggest that transport of flavonoid glucosides into barley vacuoles is specific for apigenin derivatives. In contrast, transport of hydroxyprimisulfuron-glucoside is strongly stimulated by MgATP (2.5-3 fold), not sensitive toward bafilomycin, and much less sensitive to dissipation of the ΔpH, but strongly inhibited by vanadate. Uptake of hydroxyprimisulfuron-glucoside is also stimulated by MgGTP or MgUTP by about 2-fold. Transport of both substrates is not stimulated by ATP or Mg2+ alone, ADP, or the nonhydrolyzable ATP analogue 5′-adenylyl-β,γ-imidodiphosphate. Our results suggest that different uptake mechanisms exist in the vacuolar membrane, a ΔpH-dependent uptake mechanism for specific endogenous flavonoid-glucosides, and a directly energized mechanism for abiotic glucosides, which appears to be the main transport system for these substrates. The herbicide glucoside may therefore be transported by an additional member of the ABC transporters.

In plants, secondary products such as species-specific phenolics as well as foreign compounds are often glycosylated in order to increase their water solubility. Storage of these compounds within the large central vacuole, a compartment with a very low metabolic activity, is generally assumed to protect the plant cell against potentially toxic effects of these substances, especially of their non-glycosidic form. Corresponding processes exist for the excretion of toxic conjugates in animals, e.g. at the canalicular membrane of liver hepatocytes (1,2).
Transport mechanisms of some alkaloids and phenylpropane derivatives have been suggested to be species-and substratespecific (3)(4)(5). However, vacuolar storage of glucosylated abiotic substances such as 2,4-dichlorophenoxyacetic acid or transport of hydroxyprimisulfuron-glucoside as well as uptake of phenolic compounds not present in the considered species into vacuoles have been demonstrated (6 -8). These observations indicate that the vacuole has the potential to detoxify and store not only endogenous but also foreign, biotic, and abiotic glucosylated substances.
Besides glycosylation, further metabolic modifications of the basic C 15 structure of flavonoids are well known and include oxidation, hydroxylation, methylation, and acylation reactions (9), leading to the giant chemical diversity within this class of natural phenolic compounds. The respective ligands may play a role in substrate recognition of the corresponding, species-specific, vacuolar transporter. In the case of Petroselinum hortense and Daucus carota cell cultures, it has been shown that acylation of apigenin 7-O glucoside and an anthocyanin, respectively, was a prerequisite for uptake into vacuoles (5,10) and that protonophores inhibited uptake of the anthocyanin into Daucus vacuoles (5). Different strategies may be involved in vacuolar accumulation of coumaric acid glucosides: (i) vacuolar uptake of o-coumaric acid glucoside occurring in Melilotus alba was shown to be independent of ATP, but involved an isomerization of the trans-to the vacuolar deposited cis-isomer (11). (ii) Esculin, a coumaric acid glucoside occurring in, e.g. potato but not in barley, was transported into barley vacuoles in an ATP-dependent, protonophore-sensitive manner, suggesting the existence of a proton antiport mechanism (7).
Recently, Marrs et al. (12) demonstrated that the gene characterized by the bronze-2 mutation, which is the last genetically defined step in anthocyanin biosynthesis in maize, encodes a glutathione S-transferase. They provided evidence that vacuolar deposition of the maize anthocyanin occurs via an intermediary, but yet unidentified, glutathione conjugate. From previous results showing the presence of an energized glutathione-conjugate ATPase (13,14), it was suggested that anthocyanins may be accumulated within the vacuole via such a pump.
Little information is available on the transport of glucosylated xenobiotics. We have shown that the herbicide glucoside hydroxyprimisulfuron-glucoside is transported into vacuoles of barley and that this transport is stimulated by safeners, substances conferring tolerance toward herbicides (8). However, the mechanism of this transport has not been characterized yet.
Vacuolar accumulation of different compounds can be driven by the H ϩ -ATPase or H ϩ -pyrophosphatase via an H ϩ antiport mechanism (e.g. Na ϩ /H ϩ , Ca 2ϩ /H ϩ ), as a response to ⌬⌿ (e.g. malate) (15) or directly by ATP as demonstrated for glutathione-conjugates (13,14) and bile acids (16). Calculations based on ⌬G 0 values of ATP hydrolysis assuming cytosolic ATP, ADP, and P i concentrations as published demonstrate that a much higher vacuolar accumulation can be expected by a directly energized glutathione-conjugate pump compared with secondary energized transport mechanisms (17).
There are many open questions concerning the energization of glucosylated phenolic substances, and no information is available on the transport of glucosylated xenobiotics. Therefore it was of interest to investigate (i) whether species-specific and/or general vacuolar transport mechanisms exist for endogenous and foreign glucosides in the same plant and (ii) which type of energization is used by the respective transport system.

EXPERIMENTAL PROCEDURES
Barley (Hordeum vulgare L. cv. Bakara) was grown as described (8). Mesophyll cell protoplasts and vacuoles were isolated from 10-day-old primary leaves following a procedure of Rentsch and Martinoia (18).
Uptake  Unless stated otherwise, uptake studies with unlabeled isovitexin were performed with 0.1 mM isovitexin in the above mentioned incubation medium with six tubes per condition and time point. After centrifugation the aqueous supernatants of three tubes were pooled, lyophilized, and redissolved in 100 -200 l of 80% (v/v) methanol before HPLC analysis. Separation of barley flavonoids was performed on the Shimadzu HPLC system using a Nucleosil RP-18 column (125 mm ϫ 4.6 mm; 5 m grain size; CS Chromatographie, Langerwehe, Germany) and a gradient system described previously (21). Uptake was calculated based on the vacuolar content of the major flavonoid saponarin showing no change in its concentration during the time course of the experiments (see "Results") or based on the 3 H 2 O counts measured in separate assays. Unless stated otherwise, uptake rates of hydroxyprimisulfuron-[ 14 C]glucoside were calculated by subtracting the radioactivity measured after 2 min of incubation from the 20-min values and uptake rates of unlabeled isovitexin by subtracting the vacuolar isovitexin/saponarin ratio after 2 min from the corresponding 15-min value. K m and V max values were calculated using a computer program (Enzfitter, Biosoft, Cambridge, United Kingdom).

RESULTS
Plants contain a variety of glycosylated endogenous compounds and are able to glucosylate abiotic substances as a possible step in the detoxification of xenobiotics. For both groups of glucosides vacuolar localization is generally assumed.
In an attempt to compare the transport specificities of an abiotic glucoside with two plant-borne glucosides across the vacuolar membrane, we used hydroxyprimisulfuron-[ 14  texin uptake was studied using unlabeled substrate and detection of the flavonoid by HPLC. Vacuolar and whole leaf extracts of Hordeum vulgare L. cv. Bakara contain only trace amounts of isovitexin being the precursor of saponarin (isovitexin 7-Oglucoside) (22), which accumulates as the major compound (Fig. 2B). Unlabeled isovitexin is readily taken up by barley vacuoles with a time dependence and MgATP stimulation comparable with the results obtained using [ 14 C]isovitexin. The fact that only the isovitexin peak increases with time ( Fig. 2B), while the amounts of all other detected compounds remain constant, suggests that neither isovitexin is converted into saponarin via glucosylation nor saponarin is deglucosylated to isovitexin during the course of the experiment. This observation is further supported by the fact that the uptake rate did not change when barley vacuoles were incubated in the presence of 100 M isovitexin together with 10 mM UDP-glucose (Table I). The saponarin content (0.42 Ϯ 0.09 ϫ 10 Ϫ3 mol ϫ liter Ϫ1 vacuolar volume, mean of 10 determinations) can therefore be used as a marker for vacuolar space in isovitexin uptake experiments in addition to 3 H 2 O volume determined in separate assays. The transport rates calculated using the two parameters proved to be identical within experimental error.
Uptake of the abiotic hydroxyprimisulfuron-[ 14 C]glucoside (Fig. 1C) by barley mesophyll vacuoles has been shown to be linear for at least 20 min in a previous publication (8).
Transport of isovitexin is a saturable process with an apparent K m value of 82 Ϯ 38 M and V max of 6.09 Ϯ 1.66 ϫ 10 Ϫ7 mol of isovitexin ϫ s Ϫ1 ϫ liter Ϫ1 (n ϭ 5) (data not shown). In contrast, the hydroxyprimisulfuron-[ 14 C]glucoside exhibts a linear concentration dependence up to 250 M (data not shown), indicating the presence of a low affinity transport system.
ATP-dependent stimulation of the herbicide glucoside and the flavonoid glucosides is remarkably different: while isovitexin uptake is stimulated 1.4-fold, a 2.5-3-fold increase of the uptake rate can be observed for the herbicide glucoside (Table  I). However, the MgATP-stimulated transport rates of hydroxyprimisulfuron-glucoside and isovitexin (4.32 Ϯ 1.21 ϫ   Ͼ 7), demonstrating uptake against the concentration gradient. Mg 2ϩ or ATP alone as well as ADP are not able to stimulate uptake of both subtrates, and the nonhydrolyzable ATP analogue AMP-PNP cannot substitute for ATP, suggesting that ATP hydrolysis is a prerequisite for transport energization (Table I). In the case of hydroxyprimisulfuron-glucoside, but not for isovitexin, UTP and GTP can partially substitute for ATP, stimulating uptake about 2-fold. Pyrophosphate, driving the proton pumping pyrophosphatase, does not stimulate the uptake of both glucosides. However, the activity of the pyrophosphatase amounts only to 25% of the activity of the vacuolar H ϩ -ATPase in 10-day-old primary leaves of barley (data not shown).
Differences in the transport mechanisms of the two glucosides can also be observed using inhibitors of ATPases and reagents affecting ⌬pH or ⌬ (Table II). MgATP-stimulated transport of the herbicide glucoside is not inhibited by bafilomycin A1, a highly specific inhibitor of the vacuolar membrane H ϩ -ATPase (23). The uptake of isovitexin decreases in the presence of this antibiotic to levels observed in the absence of ATP. Vanadate, an inhibitor acting as a phosphate analogue, strongly decreases the uptake of hydroxyprimisulfuron-glucoside, but not of isovitexin. The transport of both glucosides is not inhibited by azide, an effector of F-type ATPases. A different behavior of the two transport systems is also observed after the addition of NH 4 Cl, which dissipates the ⌬pH across the membrane, resulting in a strongly inhibited isovitexin transport, whereas hydroxyprimisulfuron-glucoside is only slightly affected in the presence of MgATP. However, addition of NH 4 Cl in the absence of MgATP reduces the uptake rate of the herbicide glucoside to about 44% of the control without ATP. Thus, a nonenergized component of herbicide glucoside uptake may be affected by protonophores in contrast to the major component observed in the presence of ATP. Valinomycin, a K ϩspecific ionophore, has an inhibitory but yet weaker effect on flavonoid glucoside uptake compared with the effect of the ⌬pH-dissipating reagent. Surprisingly, saponarin efflux is not observed after destroying ⌬pH or ⌬⌿ (data not shown).
To obtain further information on substrate specificities of the two transport systems, competition experiments were performed (Table III). As unstimulated uptake of hydroxyprimisulfuron-glucoside is very low (Table I), MgATP was added in competition experiments with the herbicide glucoside. In contrast, the isovitexin competition experiments were performed in the absence of MgATP with only a 3-fold excess of all competitors (120 M) due to the limited solubility of flavonoids in aqueous solution. However, the degree of inhibition did not change in the presence of ATP (data not shown). Uptake of the apigenin-derivative isovitexin is reduced or completely inhibited in the presence of other apigenins (Table III). On the other hand, luteolin derivatives do not affect isovitexin uptake. Isovitexin uptake is reduced to about 40% by the addition of ␤-methylumbelliferyl glucoside, while the corresponding ␣-isomer has no effect. All other phenolic glucosides tested so far show only low competition with isovitexin uptake. It should be noted that addition of the flavonoid glucosides or other glucosides competing isovitexin did not result in changes of the HPLC pattern. Accordingly, no additional peaks were detected at 340 nm, suggesting that chemical reactions or interactions of competitors with isovitexin are unlikely.
Surprisingly, the competition pattern of apigenin glucosides added to hydroxyprimisulfuron-glucoside at a final concentration of 120 M is comparable with the results obtained with isovitexin (Table II) . 100% correspond to uptake rates given in Table I. Values are means of three to five independent experiments if S.D. are given or from two experiments (without S.D.), each consisting of five and two replicates for hydroxyprimisulfuron-glucoside and isovitexin uptake, respectively. For calculation of uptake rates see the legend to Table I III  Influence of potential competitive inhibitors, flavonoid-glucosides, and other glucosylated substances, on the uptake of isovitexin and hydroxyprimisulfuron-glucoside Isolated vacuoles were incubated in the presence of 40 M HPSglucose or isovitexin and further compounds as indicated. Due to the limited solubility of phenolic substances in aqueous solution, external concentrations of the glucosides were 41 M for vitexin and 120 M for all other substances. 100% correspond to an uptake rate of 2.43 Ϯ 0.36 ϫ 10 Ϫ7 mol of isovitexin ϫ s Ϫ1 ϫ liter Ϫ1 and 1.82 Ϯ 0.41 ϫ 10 Ϫ8 mol of HPS-glucose ϫ s Ϫ1 ϫ liter Ϫ1 . Uptake experiments with isovitexin were performed in the presence of 1 mM Mg 2ϩ . In competition experiments with HPS-glucose, flavonoid competitors were added at 120 M while all other glucosides were added at 1 mM, since preliminary experiments with 120 M showed no inhibition. Uptake of the HPSglucose was measured in the presence of 3 mM ATP and 4 mM Mg 2ϩ . Values are means of 2 (no S.D. values) to 3 (S.D. values given) independent experiments. Each experiment consists of five replicates for hydroxyprimisulfuron-glucoside and two replicates for isovitexin uptake. For calculation of uptake rates see the legend to Table I take of hydroxyprimisulfuron-glucoside to about 80%, while vitexin has a much lower effect. In addition, isovitexin itself reduces uptake of the herbicide glucoside by about 55%. Other glucosides, which were in contrast to isovitexin experiments added at a final concentration of 1 mM, inhibit transport of the abiotic compound between 5 and 40% with esculin being the most inhibitory. In correspondence to the results obtained for isovitexin, the ␤-isomer of methylumbelliferyl glucoside has a more pronounced inhibitory effect on the uptake of hydroxyprimisulfuron-glucoside than the isomer bearing the glucose residue in the ␣-position. However, ␤-methylumbelliferyl glucoside decreases the transport of the herbicide glucoside only by about 30%, while isovitexin uptake is stronger affected by this compound (60% inhibition).

DISCUSSION
Discrepancies exist between the observation that investigations using plant-specific secondary products exhibited speciesspecific uptake into vacuoles of the corresponding substance and the description of heterologue or even abiotic substances entering the vacuole (3-5, 7, 8). The goal of our investigation was to elucidate whether two different glucosides taken up by barley vacuoles use identical or different transport systems and which energization mechanisms are involved.
Our results strongly suggest that two different mechanisms exist for the transport of the flavonoid glucoside isovitexin (apigenin 6-C-glucoside, Fig. 1A) and of a herbicide glucoside (hydroxyprimisulfuron-glucoside, Fig. 1C) for the following reasons: (i) uptake of hydroxyprimisulfuron-glucoside is quite slow under nonenergized conditions but is stimulated 2.5-3-fold in the presence of MgATP. In contrast, isovitexin uptake exhibits a much higher absolute uptake rate, while the addition of MgATP has only a slight but reproducible effect ( Fig. 2B and Table I). (ii) There are remarkable differences in nucleotide specificities, since UTP and GTP can partially substitute for ATP only in the case of the herbicide glucoside (Table I). The observation that GTP rather inhibits flavonoid uptake is not understood yet. (iii) Transport of isovitexin and hydroxyprimisulfuron-glucoside is differentially affected by inhibitors specific for different types of ATPases.
Based on these observations we propose that two glucoside transporters exist differing in their energization mechanism. Isovitexin transport by barley vacuoles is driven by the ⌬pH, while at least 60% of hydroxyprimisulfuron-glucoside uptake is mediated by a directly energized carrier.
The herbicide glucoside carrier may be a novel member of the transport ATPases belonging to the ABC family, which have been found during the last years in animals, fungi, and plants. ABC transporters are thought to be mainly involved in the cellular detoxification mechanisms (24). However, for many of these ABC transporters only the respective gene is known, while the corresponding substrate is unknown (25). For plants it has been shown that two different transport activities reside in the vacuolar membrane, which are directly energized by ATP: the transfer of glutathione S-conjugates (13,14) and of bile acids (16). These carriers accumulate their substrate in the vacuole, even if the vacuolar H ϩ -ATPase is inhibited by bafilomycin and the ⌬pH abolished by NH 4 Cl. In the case of hydroxyprimisulfuron-glucoside, the uptake rates were too low to proof accumulation against a concentration gradient. However, the general properties are very similar as compared with the other described directly energized transporters: requirement for MgATP; GTP and UTP can partially substitute for ATP; transport is not inhibited by bafilomycin and NH 4 Cl but by vanadate. Therefore we suggest that the MgATP-dependent component of the hydroxyprimisulfuron-glucoside transport activity occurs by a directly energized ATPase.
So far, transport of species-specific flavonoids across the vacuolar membrane has only been studied for acylated flavonoid glycosides (5,10). In these cases, the corresponding deacylated compounds were not taken up by the vacuoles. In this report, we clearly show that neither acylation nor conjugation to glutathione as proposed by Marrs et al. (12) for maize anthocyanins are necessary for vacuolar transport and accumulation of flavonoid-glucosides in barley.
The fact that treatment with NH 4 Cl inhibits isovitexin uptake into barley vacuoles strongly suggests that flavonoid-glucoside import may occur via an H ϩ /isovitexin antiport mechanism. Attempts to demonstrate a proton extrusion induced by isovitexin using intact vacuoles or tonoplast vesicles and the pH-sensitive fluorescent dye 9-amino-6-chloro-2-methoxyacridine, as recently shown by Getz and Klein (26) for the sucrose/ H ϩ -antiporter of red beet storage parenchyma, failed due to chemical interaction of the dye with the flavonoid. However, as the isovitexin uptake rate is also reduced in the presence of valinomycin, a K ϩ -ionophore known to dissipate the membrane potential, flavonoid glucoside transport may also be coupled to that of K ϩ . An alternative explanation would be that the carrier is activated at inside positive membrane potentials generated by the vacuolar proton pumps (27).
Saponarin, the major barley flavonoid, is synthesized by a soluble UDP-glucose-dependent glucosyltransferase (Table I; Ref. 28). The fact that isovitexin is taken up efficiently by isolated vacuoles suggests that in vivo the glucosylation step is faster and/or that the affinity of of the transporter for saponarin is much higher than for isovitexin.
Other apigenin derivatives inhibit isovitexin uptake in contrast to luteolin-type compounds as lutonarin and orientin (Table III, Fig. 1B), and the luteolin derivative orientin is not taken up at all by barley vacuoles (Fig. 2A). As apigenin and luteolin differ only in the molecular structure of the aromatic B-ring (apigenin with a single hydroxyl group in 3Ј-position, luteolin with two hyxdroxyl groups in positions 3Ј and 4Ј), the substitution pattern of the B-ring seems to contain an important recognition signal for binding and transport of the apigenin derivative. However, ␤-methylumbelliferyl glucoside inhibits isovitexin uptake much stronger than the ␣-isomer, indicating that (i) the position of the glucose substituent plays an important role in substrate binding and (ii) that other structural features than the B-ring may be additionally responsible for recognition.
Surprisingly, inhibition of hydroxyprimisulfuron-glucoside transport with different glucosides shows a similar picture as compared with isovitexin. Therefore, it is suggested that the substrate recognition is comparable for both transport systems and that in the case of isovitexin a directly energized component is not apparent due to the much higher uptake activity of the ⌬pH-dependent system.
Our results presented here indicate that at least two different carriers are responsible for the transport of glucosylated substances across the vacuolar membrane. The one is involved in detoxification and storage processes of endogenous phenolic compounds. Concerning the directly energized carrier system, the interesting questions arises, whether the directly energized transport system drives the uptake of an endogenous, so far unknown glucoside and whether plants have evolved a general internal "excretion" system for exogenous compounds that are recognized as potentially toxic by the plant.