Proton-Peptide Co-Transport in Broad Bean leaf Tissues - NCBI

Plant Physiol. (1994) 106: 1023-1031

Proton-Peptide Co-Transport in Broad Bean leaf Tissues Aziz Jamai, Jean-François Chollet, and Serge Delrot* Laboratoire de Physiologie et Biochimie Végétales (A.J., S.D.), and Laboratoire de Synthèse Organique et Organométallique (J.-F.C.), Unité Associée Centre National de la Recherche Scientifique 574, Université de Poitiers, 25 Rue du Faubourg Saint-Cyprien, 86000 Poitiers, France

ture, including several antibiotics (Dantzig et al., 1992), aspartame (Haque and Mozaffar, 1993), anticancerous agents (Hori et al., 1993), and HIV-1 protease inhibitors (Ghosh et al., 1993). The di/tripeptide transporter of Lactococcus lactis has recently been characterized as a proton symporter (Smid et al., 1989) and is quite different from another transporter mediating the uptake of oligopeptides larger than four amino acid residues (Kunji et al., 1993). In Bacillus subtilis, the dipeptide transporter is rapidly induced as cells enter stationary phase and initiate sporulation (Slack et al., 1993). This bacterium also produces bacilysin, a dipeptide antibiotic that is transported by the dipeptide transporter of Staphylococcus nureus (Perry and Abraham, 1979). The dipeptide permease of Salmonella typhimurium (Elliott, 1993) and Escherichia coli (Verkamp et al., 1993) also mediates the transport of 5aminolevulinic acid, a precursor of tetrapyrrole synthesis. Yeasts also contain a dipeptide transporter that is distinct from the amino acid transporters (Marder et al., 1977). Despite many early reports of peptide-like compounds in plant tissues, the role of these compounds is still poorly understood (Higgins and Payne, 1989). In addition to their possible nutritional role in plants, small peptides, including di- and tripeptides, may be important in plant-pathogen interactions and as compounds affecting the taste and aroma of various foods (Higgins and Payne, 1989). In higher plants, peptide uptake has been characterized only in the scutellum of barley and other cereals (Sopanen et al., 1977, 1978; Sopanen, 1979; Higgins and Payne, 1977, 1978; Walker-Smith and Payne, 1984a, 1984b; Salmenkallio and Sopanen, 1989). During the germination of the grain, protein hydrolysis occurs in the starchy endosperm, and the resulting peptides and amino acids are transferred to the embryo via the scutellum, a modified cotyledon that is differentiated as an absorptive structure. In the endosperm, protein hydrolysis is not complete because the carboxypeptidases of the endosperm do not act on di- and tripeptides, and no other peptidases are present. On the contrary, the scutellum contains high activities of peptidases that may hydrolyze the small peptides (Sopanen et al., 1977). In barley grains, the peptide transport system rapidly develops in the scutellum at the beginning of germination (Sopanen, 1979). The absorption of peptides is at least

The transport of [‘‘C]glycyl-glycine (Cly-Cly) has been characterized in leaf discs from mature exporting leaves of broad bean (Vicia faba 1.). In terms of glycine (Cly) equivalents, the rate of transport of Cly-Cly was similar to that of Cly uptake. Uptake of Cly-Cly was localized mainly in the mesophyll cells, with little accumulation in the veins. It was optimal at pH 6.0, sensitive to thiol reagents and metabolic inhibitors, and exhibited a single saturable phase with an apparent Michaelis constant of 16 mM. Cly-Cly did not inhibit the uptake of labeled Cly. Addition of ClyCly induced a concentration-dependent pH rise in the medium, showing that peptide uptake i s mediated with proton co-transport. Cly-Gly also induced a concentration-dependenttransmembrane depolarization of mesophyll cells with an apparent Michaelis constant of 15 mM. This depolarization was followed by a transient hyperpolarization. When present at a 10-fold excess, various peptides and tripeptides were able to inhibit Cly-Cly uptake with the following decreasing order of efficiency: Cly-Cly-Cly = leucineCly > Cly-tyrosine > Cly-glutamine = Cly-glutamic acid > Clyphenylalanine > Cly-threonine > Cly-aspartic acid = Cly-asparagine = aspartic acid-Cly. Cly inhibited the uptake of Cly-Gly only slightly, whereas tetraCly and the tripeptide glutathione were not inhibitory. The dipeptides inhibiting Gly-Gly uptake also induced changes in the transmembrane potential difference of mesophyll cells and were able to affect in a complex way the response normally induced by Cly-Cly. Altogether, the data demonstrate the existence of a low-affinity, broad-specificity H+/peptide co-transporter at the plasma membrane of mesophyll cells. The physiological importance of this transporter for the exchange of nitrogenous compounds in mature leaves remains to be determined, as do the details of the electrophysiologicalevents induced by the dipeptides.

In addition to amino acid transporters, the plasma membrane of prokaryotic and eukaryotic cells may contain peptide transporters that exhibit a relatively broad specificity.Peptide transport is well documented in animal cells, in yeasts, and in bacteria, but relatively few studies have dealt with the uptake of peptides in plant tissues. The brush border of the small enterocyte is a major route for the intestinal absorption of peptides, which some hydrophilic drug peptides mimic (Dantzig et al., 1992). Dipeptide and tripeptide uptake is much more efficient than amino acid uptake for the retrieval of products of protein hydrolysis from the gut lumen (Matthews, 1983). The transporter responsible for this is also of particular interest because numerous pharmaceuticals are dipeptides or possess a dipeptide-like struc-

Abbreviations: Gly-Gly, glycyl-glycine; NEM, N-ethylmaleimide; pCMBS, para-chloromercuribenzenesulphonicacid; PD, transmembrane potential difference.

* Corresponding author; fax 33-49-55-93-74. 1023

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Plant Physiol. Vol. 106, 1994

as important as the absorption of amino acids for the nitrogen nutrition of the embryo (Higgins and'Payne, 1978, 1981). The peptide transporter of the scutellum may transport peptides of up to four residues (Sopanen et al., 1977; Higgins and Payne, 1978), obeys Michaelis-Menten kinetics (Higgins and Payne, 1977; Sopanen et al., 1977, 1978), and is stereospecific, since it discriminates against peptides containing Damino acids regardless of their position in the peptide (Higgins and Payne, 1978). Peptide transport in germinating barley occurs against a concentration gradient and is inhibited by dinitrophenol, sodium azide, anoxia, and acetate, which have been taken as evidence for a proton-dependent uptake (Higgins and Payne, 1977). The transporter is inhibited by the thiol reagents NEM and pCMBS and by phenyl arsine oxide, a reagent specific for vicinal dithiols (Walker-Smith and Payne, 1984b). Evidence was obtained that suggests the presence of two components for the peptide uptake system in barley scutellum (Hardy and Payne, 1991). Although peptide transport contributes beyond a doubt to the nutrition of the cereal embryo, one may wonder what becomes of this system when the plant becomes autotrophic. In most of the higher plants, inorganic nitrogen absorbed by the roots is transported to the leaves by the xylem sap. After reductive assimilation resulting in amino acid biosynthesis in the leaves, amino acids are the predominant form of nitrogen available for transport. Because of the change in the form of reduced nitrogen (amino acid in autotrophic tissue versus peptide in heterotrophic tissue), the strong activity of various amino acid transporters (Bush, 1993)in the mature leaf might result in the disappearance of the peptide transporter, given the rapid tumover of plasma membrane proteins. Therefore, the present work was conducted to determine whether the mature leaf tissues are also able to take up peptides. We also characterized the energetics of this uptake system because the mechanism of transport has not been investigated in detail. The experiments were run with the dipeptide Gly-Gly, because it may be relatively easily synthesized and because Gly transport has already been extensively characterized in broad bean (Viciafabu L.) leaf tissues (Despeghel, 1981), thus allowing comparisons between Gly and Gly-Gly transport.

diketopiperazine (Fischer, 1906), followed by the, partia1 hydrolysis of the latter product to yield Gly-Gly (Fischer, 1905). Hydrolysis can be conducted in an acid or alkaline medium, but alkaline hydrolysis was preferred because it c:an be more easily controlled. Gly ethyl ester hydrochloride (0.02 mol; 2.79 g) was dissolved in 5 mL of ethanokwater (2:98) containing 18.5 MBq [l-'4C]Gly ester hydrochloride (2035 MBq mmol-I, Isotopchim, Ganagobie, France). After cooling to O to 5OC, triethylamine (0.02 mol, 2.8 mL) was added and stined at a rate such that the temperature remained below 5OC. ' m e mixture was allowed to stand at room temperature for 48 h. After cooling to 4OC for 4 h, the precipitate was isolated by filtration over a sintered glass filter funnel. The crystalline product was then washed with cold water (1 mL) and ccdd absolute ethanol (3 mL). After drylng ovemight in vamo, the Gly anhydride was obtained (0.63 g, 5.52 mmol, 55%). This compound was dissolved in aqueous sodium hytiroxide (1 N NaOH, 6.3 mL) and the mixture was allowed to stand at room temperature for 20 min. Hydrochloric acid was then added (1 N HCl, 6.3 mL) and water was eliminated with a rotary evaporator. After drylng over P205 in a vacuum desiccator, ['4C]Gly-Gly was obtained with sodium chloride in a quantitative yield (66.45% Gly-Gly and 33.55% NaC1). To remove NaC1, the crude product was suspended in water (0.5 mL) and filtered. The crystalline product was washed on the filter with cold water (0.5 mL) and absolute ethanol(l0 mL). Pure Gly-Gly was then obtained (yield = 65%). f i e specific NMR spectra were activity was about 1.84 MBq "01-I. recorded in deuterium oxide with a JEOL EX 90 spectrometer at 89.5 MHz ('H) and 22.5 MHz (13C)using 3-trimethylsilylpropionic acid-d4-sodium salt as intemal standard. The abbreviation used is s (singlet).The data were as follows: NMR 'H (dppm, D20):3.83 (s, 2H, CH2); 3.91 (s, 2H, CH,); 4.86 (s, 4H, NH,, NH, COOH); NMR I3C(dppm, DzO): 43.4 (CH,); 169.9 (COOH); 179.3 (CO).

MATERIALS A N D METHODS

The lower leaf epidermis was peeled with fine forceps, and leaf discs (20 mg fresh weight each) were punched with a 12m"mdiameter cork borer. Unless stated otherwise, the peeled discs were floated for 30 min on a solution containing 20 m~ Mes/NaOH (pH 6.0), 250 m~ mannitol, O.!i m~ CaC12, and 0.25 m~ MgC12.The discs (12 discs for 8 mL of medium) were then transferred for various times, usually 30 min, on the same medium containing 1m~ [I4C]Gly-Gly(final specific activity of the medium: 9.1 MBq mL-', 1.84 MEIq "01-I). Incubation was run under mild agitation on íi reciproca1 shaker at room temperature. At the end of inaibation, the discs were rinsed (3 X 2 min) in preincubation medium to remove the apoplastic label. Depending on the experiments, the discs were either lyophilized for autoradiography or processed for liquid scintillation counting (Sakr et al., 1993). For autoradiography, lyophilized leaf discs were exposed for 10 d on Hyperfilm p-max films (Amersham, Les IJlis, France) at room temperature.

Plant Material

Nonnodulated broad bean (Vicia fuba L. cv Aguadulce) were grown on venniculite in a controlled environment under the following conditions: 16 h of light (14 W m-', Sylvania tubes F 65 W Gro-Lux) at 22 f l 0 C with a RH of 65 f 5% throughout. The plants were watered daily with Hoagland solution and used for experiments when they were about 3 weeks old and possessed four or five expanded bifoliate leaves. The experiments were run with these mature, exporting leaves. Synthesis of ["CIGly-Gly

Since ["CIGly-Gly was not commercially available, this compound was synthesized by the diketopiperazine method, which offers the advantage of giving relatively good yields in a few steps. This method involves the synthesis of 2,5-

Uptake Experiments

Peptide Transporter of the Plasma Membrane

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Metabolism of ['4C]Cly-Cly

After uptake of 1 mM [I4C]Gly-Glyfor 30 min, leaf discs were extracted in 5 N acetic acid at 100°C for 20 min. The extracts were purified and dansylated as described by Higgins and Payne (1981). Unlabeled Gly and Gly-Gly (standards) were dansylated and separated by TLC on polyamide sheets (Higgins and Payne, 1977) using acetic acid:toluene (90:10, v/v) as the solvent system. This chromatography completely separated dansylated Gly-Gly (immobile) and dansylated Gly (mobile). The radioactive dansylated extracts were separated in the same way and autoradiographed for 15 d. The position of the radioactive spots was compared with the position of the fluorescent standards. O

pH Measurements

pH changes in the medium were recorded as described by Despeghel and Delrot (1983). Mature leaf tissues (0.5 g) without lower epidermiswere incubated on 20 mL of medium P, containing 250 m~ mannitol, 0.5 m CaC12, and 0.25 m~ MgCl, (initial pH 6.0). The incubation solution was continuously mixed with a rotary stirrer, and the pH of the medium was monitored with a pH recording unit (Radiometer, Copenhagen, Denmark), consisting of K4040 (calomel) and G2040 C (glass) electrodes. ElectrophysiologicalMeasurements

The ID ' of mesophyll cells was measured by standard electrophysiological techniques as described by MBatchi et al. (1986). Briefly, a peeled leaf fragment was stuck (peeled side up) to the bottom of a Petri dish with an inert paste. The tissues were rinsed and then incubated with medium P (described above) and buffered at pH 5.0 with 20 m~ Mes/ NaOH. A glass micropipette (tip diameter = 1 pm, tip resistance 5 to 20 Mil, filled with 3 M KCI) was inserted into a mesophyll cell with a mechanical micromanipulator. This electrode and a reference electrode filled with 3 M KC1 in 1% agar and immersed in the medium were connected to an electrometer-amplifier (WP Instruments, New Haven, CT). The output signal of the electrometer was recorded with an oscilloscope and a chart recorder. Peptides were added from stock solutions (250, 500, or 1000 m, depending on their solubility) prepared in medium P and buffered at pH 5.0. RESULTS Uptake and Metabolism of ['4C]Cly-Gly

Mature broad bean leaf tissues took up label from a I - m ~ or from a 4 0 - m ~['4C]Gly-Gly solution at a linear rate for at least 180 min (Fig. 1).The tissues took up only 10-fold more Gly-Gly from a 4 0 - m ~solution than from a I - m ~ solution. The fact that little or no label remained associated with the discs for short incubation times (5 s ) shows the efficiency of the rinsing procedure for removal of apoplastic label. For subsequent experiments, an incubation time of 30 min was chosen. After 30 min of incubation, the amount of Gly-Gly taken up from a 1 - m solution ~ at pH 6.0 was about 1 nmol cm-' (Fig. l),which corresponds to 56 nmol g-' fresh weight.

30 60 90 120 150 180 Duration of incubation (min)

Figure 1. Time course of Gly-Gly uptake into broad bean leaf discs from a 1-mM (O, left ordinate) or a 4 0 - m ~(O, right ordinate) GlyGly solution. Data are means of 18 discs (three experiments) f SE.

Under the same conditions, the tissues took up about 2 nmol Gly cm-' from a 1 - m ["C]Gly ~ solution. Uptake of Gly-Gly and uptake of Gly were therefore similar in terms of Gly equivalents. Gly-Gly uptake was optimal at pH 6.0, declined about 50% at pH 3.0, and declined about 30% at pH 8.0 (Fig. 2). Autoradiographs made from leaf discs incubated for 30 min in the presence of 1 m~ [I4C]Gly-Gly showed that the label was taken up mainly into the mesophyll cells and that the vein network was not strongly labeled (Fig. 3A). The poor labeling of the vein network is demonstrated by comparison with autoradiographs from leaf discs incubated with 1 m~ ['4C]Suc at the same specific activity as ['4C]Gly-Gly (Fig. 3B). The rate of Gly-Gly uptake were studied as a function of Gly-Gly concentration (Fig. 4). Gly-Gly uptake rate was saturable and obeyed simple kinetics (Fig. 4A). Double-reaprocal plots yielded a K, of about 16 m~ (Fig. 4B). Similar values were obtained using Eadie-Hofstee plots (data not shown). Analysis of the radioactivity extracted from leaf tissues after 30 min of incubation showed the absence of labeled Gly in the extracts, suggestingthat Gly-Gly is not hydrolyzed into Gly during uptake (data not shown). This conclusion is further confirmed by the results of other experiments (see below, and 'Discussion"). Uptake of Gly-Gly was sensitive to permeant (NEM) or slowly permeant (pCMBS) thiol reagents, to the uncoupler carbonylcyanide-meta-chlorophenylhydrazone,and to the histidyl-specific reagent diethylpyrocarbonate (Table I). Various compounds were tested for their ability to inhibit the uptake of Gly-Gly. In these experiments, the uptake of 1 m~ ['4C]Gly-Gly was challenged with 10 m~ of the test compounds. For comparison, 10 m~ Gly-Gly was also added as a "competitive" compound in one series. Various dipeptides including Gly-Phe, Gly-Gln, Gly-Glu, Leu-Gly, and Gly-Tyr were able to significantly inhibit Gly-Gly uptake (Table 11). Although the inhibition of Gly-Gly transport by Gly-Asn, Asp-Gly, Gly-Asp, and Gly-Thr was still significant, it was


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Aging of leaf discs strongly promotes the uptake of sugars and amino acids (Sakr et al., 1993). Aging is obtained by floating the peeled leaf discs for several hours on a simple medium before transferring the discs to the uptake medium containing the labeled substrate. Uptake of [14C]Gly-Gly was compared in fresh leaf discs and in leaf discs aged for 6 h before the uptake assay (30 min). The stimulation of Gly-Gly uptake was almost 4-fold after aging (3.7 ± 0.83 versus 0.96 ± 0.12 nmol Gly-Gly cm"2, in aged and fresh discs, respectively, means of 12 replicates ± SE), whereas the stimulation of Gly uptake induced by aging was less than 3-fold (6.56 ± 0.6 versus 2.39 ± 0.9 nmol Gly cm"2 in aged and fresh discs, respectively).

1.2 E

G

-

1 0.8

3 ro 0.6 Q. >. 0.4 O >. 0 0-2 I

3.0

4.0

5.0

6.0

7.0

8.0

External pH Figure 2. pH dependence of Gly-Gly uptake from a 1-rriM solution. The discs were preincubated for 20 to 30 min at the required pH before transfer to the incubation medium buffered at the same pH. The buffers used were citrate (10 mM)/phosphate (20 ITIM) for pH 3.0, 4.0, and 5.0; Mes (20 mM)/NaOH for pH 6.0; Hepes (20 mM)/ NaOH for pH 8.0 and 9.0. Data are means of 16 discs (two experiments) ± SE.

less marked than the inhibition due to the dipeptides previously mentioned. Interestingly, the tripeptide Gly-Gly-Gly was a strong inhibitor of Gly-Gly uptake, whereas the tetrapeptide Gly-Gly-Gly-Gly had almost no effect. The tripeptide glutathione (7-Glu-Cys-Gly) did not inhibit the uptake of Gly-Gly (Table II). Gly exerted a marginal inhibition of GlyGly uptake (Table II), but in reverse experiments where the uptake of [14C]Gly was challenged with a 10-fold excess of Gly-Gly, the dipeptide did not inhibit the uptake of the amino acid (2.60 ± 0.23 nmol Gly cm"2 in control discs versus 3.07 ± 0.3 nmol Gly cm"2 in the presence of 10 HIM Gly-Gly, means of 16 replicates ± SE).

pH Measurements

To test the possibility of H+/Gly-Gly co-transport, the pH changes induced by the addition of various dipeptides in the incubation medium were monitored. Broad bean leaf tissues strongly acidify the pH of their incubation medium due to the activity of the plasma membrane pumping ATPase (Despeghel and Delrot, 1983). pH transients induced by the addition of sugars and amino acids are more easily observed when the tissues have acidified the medium down to pH 5.0 or below (Delrot, 1981; Despeghel and Delrot, 1983). For this reason, dipeptides were added to the tissues at pH 4.75 from

10

20

30

40

Gly-Gly (mM)

Figure 3. Autoradiographs of broad bean leaf tissues after uptake of [14C]Gly-Gly (A) or [14C]Suc (B). Peeled leaf discs were incubated for 30 min in the presence of 1 mM [I4C]Gly-Gly or [14C]Suc, rinsed, frozen, freeze-dried, and exposed for 10 d to autoradiographic films. The scale bar = 5 mm, and the background of the film is shown at the bottom of the figure.

Figure 4. Concentration dependence of Gly-Gly uptake into broad bean leaf discs. A, Michaelis-Menten kinetics; B, double-reciprocal plots. Data are means of 16 discs ± SE. The experiment was repeated twice with similar results.


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Table 1. Sensitivity of Cly-Gly uptake to various chemical reagents Peeled leaf discs were preincubated for 30 min either in t h e presence (pretreated) or in the absence (not pretreated) of the inhibitor and then incubated for 30 min in the presence of 1 mM ['4C]Gly-Gly and the indicated concentration of inhibitor. Data are presented as mean percent of one experiment (without pretreatment, 8 replicates) or two experiments (with pretreatment, 16 replicates). Gly-Cly Uptake Reagent

Without pretreatment

With pretreatment

% of control

Control 1 mMNEM 0.5 mM pCMBS 1 mM pCM6S 10 mM diethylpyrocarbonate 5 p~ carbonylcyanide-m-chlorophenyl hydrazone 1 mM HgCI2 a Control uptake: 1.21 f 0.10 nmol Not determined.

a stock solution adjusted to pH 4.75. Upon addition of small Gly-Gly concentrations (1.5 m, final concentration in the medium), the rate of acidification decreased (Fig. 5A). For higher concentrations (2.5 mM), a transient rise of the pH of the medium was observed. The pH rise continued for at least 3 h after addition of 5 m Gly-Gly (Fig. 5A). No detectable lag phase for the appearance of the pH transient could be observed after addition of Gly-Gly. Dipeptides that behaved as efficient inhibitors of Gly-Gly uptake, including Gly-Phe, Gly-Gln, and Gly-Thr, were also able to induce a transient pH rise of the medium (Fig. 5B). Gly-Phe decreased or stopped the acidification of the medium only when used at 10 m final concentration (data not shown). Gly-Trp, which was an efficient competitor of GlyGly uptake (Table II), was able to induce a transient pH rise

Table II. inhibition of Cly-Cly uptake by various peptides Leaf discs were incubated for 30 min in the presence of 1 mM

['4C]Gly-Gly and 10 mM of the antagonist compound. The results are means of 16 replicates -C SE (2 experiments). Inhibitory Compound

Gly-Gly Uptake nmol c n r 2

Control Glutathione

1.07 f 0.10 1.01 f 0.12

G l y-C l y-G l y-C ly GlY

0.94 f O. 13 0.86 f 0.10 0.85 f 0.05 0.79 f 0.09 0.78 f 0.09 0.67 f 0.10 0.58 f 0.08 0.56 f 0.08 0.52 f 0.07 0.50 f 0.1 1 0.45 f 0.06 0.45 f 0.04 0.44 f 0.10

Asp-Cly Gly-Asn GI y-Asp

Cly-Gly Gly-Thr

Gly-Phe Gly-Clu GIy-G In

Gly-Tyr Leu-Gly

Gly-Cly-Gly

100"

1 OOb

63

37 77

74

ND' 69 45

53 51 51

39

ND

Control uptake: 1.03 f 0.16 nmol cm-*.

ND,

even when it was added at the relatively low final concentration of 5 mM (Fig. 5C). However, the dipeptide Gly-Asp, which inhibited Gly-Gly uptake poorly (Table 11), only blocked the acidification of the medium even when used at a final concentration of 10 or 15 m (Fig. 5C). ElectrophysiologicalMeasurements

The basal PD values found before any addition in the present study ranged between -160 and -150 mV (Fig. 6), c o n f d n g the previous mean basal values of -150 mV found in the same material (M'Batchi et al., 1986). Addition of Gly-Gly to leaf tissues induced a transient depolarization of the PD of mesophyll cells. After the addition of 5 m GlyGly (final concentration), the depolarization was maximal within 2 to 3 min and lasted about 10 min (Fig. 6A). This depolarizationwas followed by a transient hyperpolarization. The same events were recorded after the addition of 10 m Gly-Gly (Fig. 6B), except that the maximal depolarization and the duration of the depolarization (1 and 5 min, respectively) were shorter than after the addition of 5 m Gly-Gly. The hyperpolarization observed after addition of 10 m Gly-Gly was maximal about 15 min after the addition of the dipeptide, lasted about 30 min, and reached about 10 mV above the initial PD. Addition of 5 m Gly-Gly ethyl ester hydrochloride induced a stronger depolarization than Gly-Gly at the same concentration, but no hyperpolarization was observed (Fig. 6C), suggesting that the free carboxylic group of the dipeptide was required to obtain the hyperpolarization. Gly-Gly-induced depolarization and hyperpolarization were concentration dependent (Fig. 7A). The depolarization obeyed a single saturation phase with an apparent K,,, of 15 m (Fig. 7B). The hyperpolarization did not fit a straight line in double-reciprocalplots (data not shown). Several consecutiveadditions of Gly-Gly to the same preparation decreased the extent of the depolarization, whereas they induced a stronger hyperpolarization. A typical example is shown in Figure 6D. A somewhat similar pattem was


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4.6

1.5 mM

Control 4.4

15 mM

x h

4.8

JGly-Asp 15 mM Gly-Asp 10 mM

4.6

Gly-Trp

o

10

20

30

40

l/S [mM- ’) x l o 2 ] Figure 5. pH changes induced by the addition of dipeptides to a

medium containing broad bean leaf tissues. A, Concentration dependence of Cly-Cly-induced pH changes; B and C, pH changes induced by various dipeptides. Except as stated otherwise, dipeptides were added at 5 mM (final concentration)in the medium. The graphs summarize the results from experiments repeated three to five times each.

observed after two successive additions of the dipeptide GlyGlu, with the difference that the first addition of Gly-Glu did not induce a hyperpolarization, whereas the second did (Fig. 6E). The presence of 10 m Gly-Glu in the medium completely prevented the depolarization and the hyperpolarization normally induced by addition of 5 m Gly-Gly (Fig. 6E).

Figure 7. Concentration dependence of Cly-Gly-indiJcedvariations of the transmembrane potential difference. A, Depolarization and hyperpolarization as a function of final Gly-Cly concentration; B, double-reciprocal plot of Cly-Gly-induced depolarization. Data are means of five measurements for each point.

The effects of Gly and of several dipeptides on the PD were compared with the effects of Gly-Gly. Actlition of Gly at 5 r n (final ~ concentration) induced a depolarization of the PD that was stronger than the depolarization induced by Gly-Gly at the same concentration. However, unlike GlyGly, Gly never hyperpolarized the PD. Rather, an incomplete repolarization was observed after the initial depolarization

Figure 6. Dipeptide-induced variations of the

PD of mesophyll cells. Data are chart records representative of three similar experiments. Cly-Gly-EE, Cly-Gly ethyl ester hydrochloride. All additions were calculated to bring 5 mM dipeptide, except in B, where Cly-Cly was added at 10 mM, final concentration.For further details, see text.

-140 -140 -120 -100 -140 U

-160 -120 Gly-Gly 30 min -140

-100


induced by Gly (Fig. 6F). The presence of 5 mM Gly in the medium only marginally affected the events induced by addition of 5 mM Gly-Gly (Fig. 6F). Several dipeptides (including Gly-Asp and Gly-Gln) were also able to induce a depolarization and/or hyperpolarization of the PD, the amplitude of these events depending strongly on the nature of the dipeptide. For example, Gly-Asp induced a rather weak depolarization followed by a small hyperpolarization (Fig. 6G), whereas Gly-Gln induced a small depolarization followed by a strong hyperpolarization (Fig. 6H). Interestingly, Gly-Gln seemed to potentiate strongly the depolarization (Fig. 6H) normally induced by Gly-Gly (Fig. 6A) but completely suppressed the hyperpolarization (Fig. 6H) normally induced by this dipeptide (Fig. 6A). Therefore, this interaction was still different from that observed with Gly-Glu, which suppressed both the depolarization and the hyperpolarization induced by Gly-Gly (Fig. 6E).

DISCUSSION

,

Despite their potential importance in plants, peptide-like compounds and the corresponding transport systems have been paid relatively little attention and deserve greater consideration (Higgins and Payne, 1989). The importance of peptides for long-distance transport in the phloem and in the xylem is still a matter of debate (Higgins and Payne, 1989) but would require the existence of peptide transporters in the plasma membrane of leaf cells. Given that several bacterial or fungal toxins possess a peptide-like structure, information pertaining to the peptide transporters (and/or receptors) may also be relevant for the understanding of plant-pathogen interactions. Due to their broad specificity, peptide transporters could also serve as targets or transporters for xenobiotics. Peptide transport has so far been characterized only in barley grains. This process is involved in retrieval of the peptides resulting from the hydrolysis of seed proteins by the embryo (Higgins and Payne, 1977; Sopanen et al., 1977). The present work shows that a peptide transporter also exists in mature exporting leaf from a leguminous species and that it takes up peptides with proton co-transport. Evidence that peptide uptake in broad bean leaf is not mediated by an amino acid transporter comes from comparison between Gly uptake and Gly-Gly uptake, from metabolic studies, from competition experiments, from electrophysiological data, and from the differential effect of aging on Gly and Gly-Gly uptake. Gly-Gly uptake was optimal at pH 6.0 (Fig. 2), whereas Gly uptake in the same material was optimal at pH 4.0 (Despeghel, 1981). The pH optimum found in the present study is also clearly different from the pH optima found for the uptake of Gly-Gly (pH 4.5, Sopanen et al., 1979) and of the peptide Gly-sarcosine in barley embryos (pH 3.8, Higgins and Payne, 1978; pH 4.5, Sopanen, 1979). Gly-Gly uptake in the mature leaf of broad bean exhibits a single saturable phase (Fig. 4), whereas Gly uptake obeys a biphasic concentration dependence at pH 4.0 with an apparent K, of 4 and 50 mM for the low- and high-affinity phases, respectively (Despeghel, 1981). Gly-Gly seems to be less concentrated by

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the vein network of broad bean leaf (Fig. 3) than are the amino acids (Despeghel and Delrot, 1983). Although Gly uptake resulted in a marginal inhibition of Gly-Gly uptake, which might be due to some competition at the active site of the peptide transporter, Gly-Gly did not inhibit Gly uptake (Table 11). The electrophysiological responses induced by Gly on the one hand and by Gly-Gly on the other hand are clearly different. Gly induced a strong depolarization followed by a slow and incomplete recovery of the PD, whereas Gly-Gly addition resulted in a weaker depolarization followed by a hyperpolarization (Fig. 6). Finally, aging, whose stimulating effect on uptake has been shown on sugars and amino acids (Lemoine et al., 1984; Sakr et al., 1993), also promotes the uptake of Gly-Gly, but the extent of the stimulation is stronger than that measured for Gly. The peptide transporter characterized in broad bean leaf tissue exhibits a rather low affinity for Gly-Gly. The apparent K, of about 16 mM found both in uptake studies (Fig. 4) and in electrophysiological studies (Fig. 8) is higher than the apparent K, found for the uptake of Gly-Gly in barley embryos (2 mM, Sopanen et al., 1978) or for the uptake of Gly-sarcosine in the same material (8 m, Higgins and Payne, 1978; 10 mM, Sopanen, 1979). Preliminary competition experimentssuggest that the specificity of the peptide transporter from broad bean leaf is broad. All of the dipeptides tested were able to more or less inhibit Gly-Gly uptake. Various dipeptide compounds including Leu-Gly, Gly-Tyr, Gly-Gln, Gly-Glu, Gly-Phe, and GlyThr were more inhibitory for uptake of [I4C]Gly-Gly than unlabeled Gly-Gly. This transporter may also transport tripeptides containing the classical N-peptidic bounds, since Gly-Gly-Gly was the strongest inhibitor among all compounds tested. However, no inhibition was observed with the tripeptide glutathione, in which Glu is attached to Cys through its 7-carboxylic group, leaving the amino and the acarboxylic group free (Table I). The ability of this transporter to recognize peptides may be limited to tripeptides, since the tetrapeptide Gly-Gly-Gly-Gly was unable to inhibit Gly-Gly uptake. In this respect, the peptide transporter from mature broad bean leaf also differs from the peptide transporter of barley scutellum, which is able to transport Gly-Gly-Gly-Gly (Sopanen et al., 1977) and other tetrapeptides (Higgins and Payne, 1978). Detailed kinetics experiments are required to determine the number of different peptide transporters present and their specificity. Since several amino acid transporters are needed to transport the different classes of amino acids (Bush, 1993), it is likely that several &/tripeptide transporters are also needed to cope with the large number of possible diand tripeptide sequences. Indirect evidence suggests that the peptide uptake system involves two components in barley embryos (Hardy and Payne, 1991). Addition of Gly-Gly to broad bean leaf tissues induces a depolarization of the PD that exhibits the same concentration dependence as Gly-Gly uptake, with an apparent K, of about 16 m for both processes (Figs. 4 and 7). This indicates that Gly-Gly uptake is electrogenic. pH measurements show that Gly-Giy additions result in concentration-dependent pH rises of the medium (Fig. 6), which strongly argues in favor of a proton-peptide co-transport uptake mechanism. These data validate the previous hypothesis, based on indirect evidence

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Jamai et al.

from inhibitor studies, that peptide uptake was occurring with proton co-transport (Higgins and Payne, 1977; Payne and Walker-Smith, 1987). Gly-induced changes in PD were similar to those reported for L e m a gibba (Fischer and Lüttge, 1981). The depolarization was interpreted as the result of proton influx mediated by the amino acid/proton co-transporter, whereas the repolarization is due to a stimulation of the proton-extruding ATPase of the plasma membrane. Upon removal of Gly, Fischer and Lüttge (1981) observed a transient hyperpolarization that was explained as the result of continued stimulation of the ATPase activity while the proton influx due to the amino acid transport was stopped. A similar transient hyperpolarizationwas observed in our experiments after addition of Gly-Gly, without removal of the dipeptide. It is not yet clear why Gly-Gly but not Gly was able to induce a transient hyperpolarization, since both compounds carry one positive and one negative charge at pH 6.0. It can be hypothesized that Gly-Gly addition would result in a higher net influx of charges into the cell, due to Gly-Gly hydrolysis, leading to a stronger response of the ATPase to the depolarization. However, chromatographic studies showed no metabolism of Gly-Gly in our experiments. Also, even if the Gly-Gly taken up from a 5 - m ~ solution would be immediately and completely hydrolyzed into two Gly molecules inside the cytoplasm, the amount of Gly appearing in the cytoplasm would be about the same as the amount of Gly taken up by the amino acid transporter from a 5 - m solution, ~ since Gly-Gly and Gly uptake possess the same activity in terms of Gly equivalent taken up (see 'Results"). Therefore, the transient hyperpolarization noticed after addition of Gly-Gly and of other dipeptides seems specific for the dipeptides. It may result from a stimulation of the proton pumping ATPase and/or from an effect of dipeptideson ion channels. These possibilities deserve further investigation, especially with respect to the known toxic properties of fungal and bacterial peptides, and with respect to the well-known occurrence of peptide-regulatedchannels in animal cells. The existence of a H+/peptide co-transporter in mature leaf tissues raises the question of its possible physiological sigxuficance. Lack of Gly-Gly hydrolysis within 30 min of uptake does not preclude a slow hydrolysis for long incubation times. The most likely role of the di/tripeptide transporter may be nutritional, but its exact importance for nitrogen transfer in adult plants must await measurements of the peptide content of the apoplast of mature leaf, its comparison with the amino acid content, and translocation experiments with labeled peptides. Peptide uptake might be just a secondary mechanism allowing the retrieval of peptides entering the leaf apoplast via the transpiration stream and/or allowing the retrieval of peptides resulting from proteolysis of cell wall proteins or glycoproteins. Altematively, peptide transport may be a secondary function for a transporter whose primary function is still unknown. Received March 21, 1994; accepted June 30, 1994. Copyright Clearance Center: 0032-0889/94/106/1023/09.

Plant Physiol. Vol. 106, 1994 LITERATURE CITED

Bush DR (1993) Proton-coupled sugar and amino acid transporters in plants. Annu Rev Plant~PhysiolPlant Mol Biol44: 513-542

Dantzig AH, Tabas LB,{Bergin L (1992) Cefaclor uptake by the proton-dependent dipeptide transport carrier of hum an intestinal

Caco-2 cells and comparison to cephalexin uptake. l3iochim Biophys Acta 1112 167-173 Delrot S (1981) Proton fluxes associated with sugar uptake in Vicia faba leaf tissues. Plant Physiol68 701-711 Despeghel JP (1981) Etude des mécanismes de l'absorption des acides aminb neutres par les tissus foliaires et de leur accumulation dans les nervures. PhD thesis. University of Poitiers, France Despeghel JP,Delrot S (1983) Energetics of amino acid uptake by Vicia faba leaf tissue. Plant Physiol71: 1-6 Elliott T (1993) Transport of 5-aminolevulinicacid by lhe dipeptide permease in Salmonella fyphimun'um. J Bacterioll75 325-331 Fischer E (1905) Synthese von Polypeptiden. IX. Berichte 38: 605-619 Fischer E (1906) Synthese von Polypeptiden. XIV. Berichte 3 9 453-474 Fischer E, Lüttge U (1981) Membrane potential changes related to active transport of glycine in Lemna gibba G1. Plarit Physiol 6 5 1004-1008 Ghosh AK, McKee SP, Thompson WJ,Darke PL, Zupy JC (1993) Potent HN-1 protease inhibitors: stereoselective synthesis of a dipeptide mimic. J Org Chem 5 8 1025-1029 Haque ZU, Mozaffar Z (1993) Influence of a dipepti'de, aspartam, on acetylcholinesteraseactivity in the mouse brain. Eiosa Biotechno1 Biochem 57: 689-690 Hardy DJ, Payne JW (1991) Analysis of the peptide carrier in the scutellum of barley embryosby photoaffinity labelling. Planta 186 44-51 Higgins CF, Payne JW (1977) Characterization of active dipeptide transport by geminating barley embryos: effects of pH and metabolic inhibitors. Planta 136 71-76 Higgins CF, Payne JW (1978) Peptide transport by germinating barley embryos: uptake of physiological di- and 'oligopeptides. Planta 138 211-215 Higgins CF, Payne JW (1981) The peptide pools OF germinating barley grains: relation to hydrolysis and transport of storage proteins. Plant Physiol67: 785-792 Higgins CF, Payne JW (1989) Plant peptides. In D Boulter, B Parthier, eds, Nucleic Acids and Proteins in Plant3 I: Structure, Biochemistry and Physiology of Proteins. Encyclopedia of Plant Physiology, Vol 14A. Springer Verlag, Berlin, pp 4313-458 Hori R, Tomita Y, Katsura T, Yasuhara M, Inui K-I, Takano M (1993) Transport of bestatin in rat renal brush-border membrane

vesicles. Biochem Pharmacol45 1763-1768 Kunji ERS, Smid EJ, Plapp R, Poolman 8, Konings WN (1993)

Dipeptides and oligopeptides are taken up via dislinct transport mechanismsin Lactococcus lacfis. J Bacterioll75: 20!52-2059 Lemoine R, Delrot S, Auger E (1984) pH sensitization of sucrose uptake during ageing of broad bean leaf tissues. Physiol Plant 61: 571-5 76 Marder R, Becker JM,Naider F (1977) Peptide tranijport in yeast: utilization of leucine and lysine containing peptides by Saccharomyces cerevisiae. J Bacterioll31: 906-916 Matthews DM (1983) Intestinal absorption of peptides. Biochem SOC Trans 11: 808-809 MBatchi 8, El Ayadi R, Delrot S, Bonnemain JL (1986) Direct versus indirect effects of p-ch1oromercuribenzener;ulphonicacid on sucrose uptake by plant tissues: the electrophjsiological evidence. Physiol Plant 68: 391-395 Perry D, Abraham EP (1979)Transport and metabolism of bacilysin


and other peptides by suspensions of Staphylococcus aureus. J Gen Microbiolll5 213-221 Sakr S, Lemoine R, Gaillard C, Delrot S (1993)Effect of cutting on solute uptake by plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves. Plant PhysiollO3 49-58 SalmenkallioM, Sopanen T (1989) Amino acid and peptide uptake in the scutella of germinating grains of barley, wheat, rice and maize. Plant Physiol 89: 1285-1291 Slack FJ, Mueller JP, Soenshein AL (1993) Mutations that relieve nutritional repression of the Bacillus subtilis dipeptide permease operon. J Bacterioll75 4605-4614 Smid EJ, Driessen AJM, Konings WN (1989) Mechanisms and energeticsof dipeptide transport in membrane vesicles of Lactococcus lactis. J Bacterioll71: 292-298 Sopanen T (1979) Development of peptide transport activity in barley scutellum during germination. Plant Physiol64 570-574

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Sopanen T, Burston D, Matthews DM (1977) Uptake of small

peptides by the scutellum of germinating barley. FEBS Lett 7 9 4-7 Sopanen T, Burston D, Taylor E, Matthews DM (1978) Uptake of glycylglycineby the scutellum of germinating barley grain. Plant Physiol61: 630-633 Verkamp E, Backman VM, Bjornsson JM,Sol1 D, Eggerstsson G (1993) The periplasmic dipeptide permease system transports 5aminolevulinicacid in Escherichia coli. J Bacteriol 175: 1452-1456 Walker-Smith DJ, Payne JW (1984a) Characteristics of the active

transport of peptides and amino acids by germinating barley embryos. Planta 162 159-165 Walker-Smith DJ, Payne JW (1984b)Characteristics of the protein carrier of the peptide-transport system in the scutellum of germinating barley embryos. Planta 162 166-173