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Valine uptake in the tap root of sugar beet: a comparative analysis with sucrose uptake
ARTICLE IN PRESS Journal of Plant Physiology 161 (2004) 1299–1314
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Valine uptake in the tap root of sugar beet: a comparative analysis with sucrose uptake Philippe Michonneau, Gabriel Roblin, Janine Bonmort, Pierrette Fleurat-Lessard* Laboratoire de Physiologie, Biochimie et Biologie mole´culaire Ve´ge´tales, Universite´ de Poitiers, UMR CNRS 6161, 40 Avenue du Recteur Pineau, 86022 Poitiers, Cedex, France Received 27 November 2003; accepted 23 February 2004
KEYWORDS Beta vulgaris; Valine uptake; Sucrose uptake; Nutrient storage; Tap root
Summary Given the lack of data on the absorption of amino acids in the tap root of Beta vulgaris, we studied the uptake of valine and compared it with that of sucrose at the same concentration (1 mM). The uptake of both substrates shared some similar characteristics. In particular, the absorption in both cases was controlled by an active process as evidenced by the inhibitory effect of CCCP and inhibitors of ATPases (DES, DCCD, orthovanadate). Both absorptions also involved the thiol and histidyl groups of protein carriers included in the plasmalemma as shown by treatment with specific compounds (PCMBS, mersalyl, NEM) inhibiting the transport of the nutrients in tissues and in purified PMV. However, it was shown that these uptakes present major differences. Firstly, unlike sucrose uptake, valine uptake was very sensitive to transmembrane electrical potential. Indeed, hyperpolarizing treatment with FC increased valine uptake but did not modify sucrose uptake. By contrast, treatment with high concentrations of KCl, which should result in depolarization of the cells, considerably decreased valine uptake and activated sucrose uptake. Secondly, ion mobilizations were different in the two types of transport. Unlike sucrose, application of valine to tissues strongly modified the time course of Hþ influx. By contrast, sucrose uptake was controlled by Kþ involvement as shown by effects either of modulators of Kþ mobilization (LiCl, TEA) or of treatments inducing Kþ starvation from the external medium. & 2004 Elsevier GmbH. All rights reserved.
Abbreviations: CCCP, Carbonyl cyanide m-chlorophenylhydrazone; DCCD, N,N0 -Dicyclohexylcarbodiimide; Dc, Membrane electrical potential; DEPC, Diethylpyrocarbonate; DES, Diethylstilbestrol; FC, Fusicoccin; HEPES, N–[2-hydroxyethyl]piperazine-N0 –[2-ethanesulfonic acid]; MES, 2-[N-morpholino]ethanesulfonic acid; NEM, N-ethylmaleimide; PCMBS, p-Chloromercuribenzenesulfonic acid; PMF, Proton motive force; PMV, Plasma membrane vesicles; TEA, Tetraethylammonium chloride; TPPþ, Tetraphenylphosphonium bromide *Corresponding author. Fax: þ33-5-49-45-41-86. E-mail address: [email protected] (P. Fleurat-Lessard). 0176-1617/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.02.005
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Introduction During plant development, photoassimilates are exchanged between sources, suppliers of metabolites (exporting leaves), and sinks, importers of metabolites (growing or storage organs). The long distance transport of photoassimilates is controlled by the osmotic gradient established in the phloem between the source and the sinks (Delrot and Bonnemain, 1985, and references therein). The first transport of assimilates occurs from the photosynthetic leaf cells into the leaf vascular system. Sucrose transport from mesophyll tissues to sites of phloem loading has been shown to be a regulated process and does not result from simple leakage (Secor, 1987). The route for transport is through the apoplast requiring active uptake by sieve tubes and contiguous cells (Giaquinta, 1983; Fisher and Evert, 1982). More specifically, the hypothesis is that sucrose absorption into the vascular tissues is mediated by an Hþ-sucrose transport (Giaquinta, 1983), a hypothesis sustained by the results of experiments with intact tissues (Daie, 1987; Gahrtz et al., 1994). In addition, physiological data have shown that phloem loading of amino acids and sucrose is mediated by different and separate carriers, all of which are dependent on an energy-requiring mechanism (Servaites et al., 1979; Sovonick et al., 1974). In contrast to phloem loading in mature leaves, few data deal with phloem unloading in sinks such as developing leaves (Schmalstig and Geiger, 1985) or storage roots (Giaquinta, 1979). As a general rule, mechanisms regulating the unloading of nutrients, from conducting to storage tissues, are not clearly understood; the pathway may be via plasmodesmatal connections in the symplast (Schmalstig and Geiger, 1985; Ding et al., 1988) or may be apoplastic (Van Bel and Kempers, 1990). There is evidence that sucrose translocated into sugar beet root is unloaded into the free space (Stein and Willenbrink, 1976) and is taken up in an unhydrolyzed form by storage parenchyma cells via a nonsaturating carrier, thereby arguing for an apoplastic pathway (Wyse, 1979). At the cell level, absorption of sucrose and amino acids has been studied on various experimental models, leaf fragments (Cheung and Nobel, 1973; Delrot et al., 1980; Despeghel and Delrot, 1983), coleoptiles (Etherton and Rubinstein, 1978), cotyledons (Komor et al., 1977; Hutchings, 1978), roots (Chin et al., 1981), and pulvinar tissues (Racusen and Galston, 1977; Otsiogo-Oyabi and Roblin, 1985). Whether these models are relevant is questionable since the cells in normal conditions in planta behave like source cells and are thus mainly
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concerned with unloading processes. However, in these particular experimental conditions, in which tissues were isolated from normal correlations encountered in planta, it has been shown that the energy of these transports may be provided by a gradient of electrochemical potential of protons across a membrane that could be generated and maintained by a vectorial membrane-bound ATPase. Advances towards a complete understanding of transport processes can be attributed to the use of isolated membrane vesicles confirming the above-defined data (Bush, 1989, 1990; Gaillard et al., 1990). Moreover, molecular approaches have shown that nutrient transport occurs by means of specific carriers (Riesmeier et al., 1992; Sauer and Stolz, 1994; Fischer et al., 1998). As a corollary, Hþ-ATPase pumps necessary to energize nutrient transport have been purified, characterized, localized, and cloned (Sze, 1985; Harper et al., 1989; Serrano, 1989; Bouche ´-Pillon et al., 1994). Our work was focused on the study of nutrient accumulation in a major sink organ, the tap root of Beta vulgaris L. As the transport of assimilates affects plant productivity, a better partitioning rather than a high photosynthesis rate will increase the sugar beet yield. The sugar beet is a biennial plant accumulating nutrients in its tap root during the first year and using this store in the second year to form reproductive organs. Following the distribution of sugars in tap root after assimilation of 14C by old and young leaves, Haeder and Beringer (1987) showed that more sucrose was found in the centre of the storage tissue in relation to the course of the vascular bundle extending from the source leaf. The tap root has an unusual structure, since conducting tissues are organized into concentric rings produced by successive supernumerary cambial layers. Nutrients are stored in xylem parenchyma and, to a greater extent, in the large and outermost pericyclic cells (i.e. storage cells in each vascular ring). In tap roots cultivated in our conditions, the determination of sucrose content reached 8176 mg g1 fresh weight (n ¼ 3). However, this average value obtained from root tissue homogenates certainly underestimates the actual sucrose concentration in the vacuole of storage cells. Previous results obtained on nutrient transport in the tap root of red and sugar beet were concerned essentially with sucrose transport in the vacuolar compartment. Based on tissue analysis, it has been shown that a major portion of the sucrose taken up from the free space is stored in the vacuole without hydrolysis (Giaquinta, 1977; Wyse, 1979). The downhill electrochemical potential gradient across
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the tonoplast is coupled with sucrose uptake via a proton antiport system and a Kþ gradient. The Hþantiport process was confirmed by use of tonoplast vesicles isolated from beet storage tissue (Briskin et al., 1985b) in which an Hþ-ATPase sensitive to nitrate was isolated (Briskin et al., 1985a). Finally, the existence of a carrier protein involved in sucrose uptake in tonoplast was demonstrated by an immunological approach (Getz et al., 1993). The question of sucrose transport at the plasmalemma has only been reported in immature sugar beet tap roots according to a classical sucrose Hþcotransport mechanism (Lemoine et al., 1988) but no true demonstration has been afforded and investigations along these lines have not been further pursued; however, this mechanism can be expected since an Hþ-ATPase activity has been demonstrated in the plasma membrane isolated from tap root tissues in red beet (O’Neill and Spanswick, 1984; Bennett et al., 1984; Poole et al., 1984; Oleski and Bennett, 1987; Briskin and Reynolds-Niesman, 1991) and in sugar beet in the course of this work. In any event, data related to the sucrose uptake at the plasmalemma level in the tap root of the sugar beet are scarce, and characteristics of amino acid uptake are completely unknown. However, many N-compounds and amino acids in particular have been detected in this organ (Schiweck et al., 1993). It was further shown that glutamate, glutamine and aspartate are the main amino acids transported from the leaf to the root in sugar beet. However, many neutral amino acids are translocated and, among these, valine is found in the phloem sap at a concentration that is estimated to be about 3 mmol l1 (Lohaus et al., 1994). Valine was chosen in the present study because the uptake mechanism of neutral amino acids has been well characterized in many plant materials and, more precisely, the uptake of valine by beet leaf cells has been widely investigated in our laboratory, thus allowing a direct comparison with the results obtained here with those made on root cells. In this context, the main features of our work were to analyse the uptake of valine, used at a relatively low concentration (1 mmol l1), and to compare it with the uptake of sucrose, also used at the same experimental conditions. In this regard, we stress that previously, except in experiments concerning the determination of kinetic parameters of sucrose unloading (Wyse et al., 1986), high concentrations (5–100 mmol l1) of substrates were employed to study uptake mechanisms in the tap root of B. vulgaris. In the present work, we first characterized the absorption of valine by beet root tissues by
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determining uptake pattern, energetics of the process, and implication of a protein component. The data thus obtained were compared throughout this work with that observed on sucrose uptake. Secondly, using PMV, we demonstrated that valine and sucrose uptake in the tap root of B. vulgaris occurred initially at the plasmalemma level in the sink cells. Thirdly, we evidenced features differentiating valine from sucrose uptake. In particular, unlike valine uptake that shares the general properties of an Hþ-substrate co-transport, sucrose uptake was shown to be poorly related to Hþ influx and transmembrane potential but correlated to a Kþ mobilization in a way needing further investigations to be more precise.
Materials and methods Plant material Sugar beets (B. vulgaris var. aramis) were grown in a greenhouse on vermiculite under natural conditions. The plants were watered daily with Snyder and Carlson (1978) nutritive solution. Supplementary lightning was provided with Osram 58/10 fluorescent tubes, giving 30 mmol photon m2 s1 from 6.00 to 9.00 a.m. and from 7.00 to 10.00 p.m. Plants were used for experiments when they were about 2 months old and bearing ten expanded leaves.
Transport assays in vivo Responses on intact systems are complicated by several factors, including metabolic fates due to aging, wounding, and compartmentalization of the substrates. These constraints were tentatively restricted through careful experimental conditions: sampling was made in a precise zone of the root, and tissue handling duration was reduced. Tissue cylinders of 12 mm diameter were taken from the root cortex with a cork borer. These tissues were sliced with a razor blade into discs 1 mm in thickness and 0.110070.0013 g in mass. These discs were divided into four quarters of 0.026870.0030 g, to facilitate uptake of the substrates. Unless stated otherwise, the quarters were immersed for 1 h in a solution at pH 5.30, containing 20 mmol l1 MES/NaOH, 0.25 mmol l1 MgCl2, 0.5 mmol l1 CaCl2, 300 mmol l1 sorbitol (added to maintain osmotic pressure in the root cells, which have been isolated from their natural environment as a result of disc sampling). The tissues were then transferred in the same medium containing
ARTICLE IN PRESS 1302 1 mmol l1 [3H]valine (final activity 9.25 kBq ml1) and 1 mmol l1 [14C] sucrose (final activity 11 kBq ml1). Incubation (generally 30 min) was run under mild agitation on a shaker at 251C. Experiments were run in parallel on both substrates on the same set of discs to compare accurately and quantitatively the modifications noted following various treatments. At the end of the incubation, the quarters were rinsed (3 3 min) in the preincubation solution to remove the apoplastic label and were placed in scintillation vials. Digestion occurred at 551C during 24 h. in a mixture of perchloric acid (56%), H2O2 (27%), and 0.1% Triton 100 (17%). Finally, 4 ml of scintillation liquid (Ecolite TM, ICN) was added to the vials, and radioactivity was counted by liquid scintillation spectroscopy (1900 TR Packard). In order to estimate the transmembrane potential gradient in beet root tissues, TPPþ was used as probe. The beet root slices were incubated and treated exactly as described above, except that assimilates in the incubation medium were replaced with 4 mmol l1 [3H] TPPþ (final activity: 2.17 kBq ml1) (Amersham, France).
Transport assays in vitro Purified PMV were prepared according to the general method of Gallet et al. (1989) from the beet roots and used to measure uptake of valine and sucrose in various energizing conditions (Lemoine et al., 1991). Briefly, the PMV at a concentration of 15 mg protein ml1 were equilibrated in a medium containing 0.3 mol l1 sorbitol, 50 mmol l1 potassium phosphate (pH 7.5), 0.5 mmol l1 CaCl2, 0.25 mmol l1 MgCl2 and 0.5 mmol l1 DTT. The PMV were then resuspended in 50 mmol l1 sodium phosphate at pH 5.5 containing 10 mmol l1 valinomycin (condition creating DpH and DC gradients: PMV in ‘energized’ state). Valinomycin creates a DC gradient following the diffusion of Kþ outwards the vesicles in the presence of the K ionophore. In every case, 1 mmol l1 [3H] valine (final activity: 41 kBq ml1) and 1 mmol l1 [U-14C]sucrose (27 kBq ml1) were added in the medium. Uptake was initiated by rapidly mixing vesicles with the incubation medium on a vortex apparatus. During experiments, solutions and test tubes were kept in a water bath maintained at 251C. At the desired time, uptake was terminated by adding 2 ml of the used medium plus 5 mmol l1 HgCl2 and filtering the content of the test tube on a Millipore HAWP filter (pore size 0.45 mm) prewetted with 2 ml rinsing medium. The test tube was rinsed with 2 ml of medium and
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poured onto the filter. The filters were then placed in a scintillation vial, dried at 501C for 1 h and immersed in 4 ml of scintillation liquid for counting.
Hþ Fluxes measurements Tap root tissues prepared as described above were preincubated for 1 h in 15 ml of a medium containing 300 mmol l1 sorbitol, 0.25 mmol l1 MgCl2, and 0.5 mmol l1 CaCl2. The quarters were then transferred to 10 ml fresh medium. Variation of the pH of the incubation medium were read on a pH-meter (Expandomatic SS2 Beckman, Roissy, Fr) provided with a combined electrode (Futura, Beckman) and linked to a potentiometric recorder. The incubation medium was aerated with a rod stirrer (Metrohm). Substrates were added in the medium as indicated in the Fig. 7A. In a series of experiments, Hþ amount was titrated either by 5 103 N HCl or 5 103 N NaOH in 2 ml aliquot withdrawn at regular intervals (generally 30 min) from the incubation medium. This experimental value was expressed for the whole incubation volume and by taking into account the weight of fresh root tissues.
Results Characteristics of valine and sucrose uptake Tap root tissues took up labelled valine and sucrose from a 1 mmol l1 solution following three successive linear components as a function of uptake duration. The general shape of absorption did not show differences between the two substrates except that valine was absorbed in a much higher amount on a molar basis than was sucrose. In both cases, the first phase, lasting 3 h, was followed by a second phase, lasting between 3 and 12 h, which was characterized by a steeper slope. The third phase, after 12 h, differed somewhat between the two substrates. In this last step, valine uptake rate became stationary whereas sucrose uptake remained high up to 36 h (Fig. 1). This long-term uptake study is however difficult to interpret because of metabolism and compartmentation of the transported molecules. In this regard, the data suggested that valine uptake may be inhibited by internal valine content or counterbalanced by an efflux, whereas sucrose uptake may continue due to its metabolization. As a practical indication, our results indicated that a 30 min-absorption time was convenient for further studies concerning the mechanism of nutrient uptake.
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Figure 1. Typical time course of 1 mmol l1 valine and 1 mmol l1 sucrose uptake by beet root slices at pH 5.3; data are means7SE (n ¼ 15). Inset: data in the first 2 h. Experiment has been made 3 times with similar results.
Figure 2. Effect of ageing in root tissues of sugar beet on the uptake of 1 mmol l1 valine and 1 mmol l1 sucrose at pH 5.3. Uptake duration: 30 min; data are means7SE (n ¼ 30) from two independent experiments.
In the course of long-term uptake, it has been previously shown that the aging of sugar beet leaf discs considerably promotes the uptake of sugars and amino acids (Sakr et al., 1993). Aging is achieved by floating the root quarters for several hours on the incubation medium before transferring the tissues to the uptake medium containing the labelled substrates. The curves in Fig. 2 show that only a small modification in the capacity of valine and sucrose absorption was seen during the first 3 h. After this lag, uptake of both substrates increased continuously and considerably until 12 h of aging. After this period, a significant
decrease was noted. Therefore, in subsequent experiments, uptake was carried out 3 h after root tissue sampling. The uptake of both valine and sucrose were shown to be dependent on the experimental pH (Fig. 3). A clear difference appears between the two substrates since it was observed that absorption was optimal at pH 5.2 for valine and pH 6.0 for sucrose. In light of these results, further experiments were carried out using MES buffer adjusted to pH 5.3 since at this value absorption of both substrates was close to the optimum and processed near the apoplastic pH.
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Figure 3. Effect of pH and buffer on the uptake of 1 mmol l1 valine and 1 mmol l1 sucrose by beet root slices. Root tissues were preincubated 3 h in buffered medium at indicated pH and uptake lasted 30 min. Two different buffers were used: 20 mmol l1 HEPES buffer for pH 4.0, 5.2 and 8.0; 20 mmol l1 MES buffer for pH 6.0 and 7.0. Data are means7SE (n ¼ 30) from two independent experiments.
Kinetic parameters of valine and sucrose uptake Concentration dependence studies were run with valine and sucrose concentrations ranging from 0.1 to 100 mmol l1. The data enabled a saturable component up to 10 mmol l1 to be distinguished (suggesting that part of assimilate uptake is carrier mediated) and a more or less linear component at higher concentrations. Eadie–Hofstee plots (Fig. 4) illustrate these data and specify that the saturable phase was composed of two components characterized by different apparent kinetic parameters shown in Table 1.
Characterization of an active process The relative active and passive components of valine and sucrose absorptions have been characterized by the use of the protonophore CCCP. Treatment with 10 mmol l1 CCCP for 1 h showed that the passive components of valine and sucrose absorptions increased when the nutrient concentration increased (near 15–20% at 0.5 mmol l1 but 60–70% at 100 mmol l1) (Table 2). Increasing the length of the CCCP treatment (Fig. 5A and B) showed a constant residual uptake of the substrates applied at 1 mmol l1. This indicated that a weak passive component occurred both for valine and sucrose absorption, which remained constant throughout the course of the experiment. For
example, a 6 h treatment inhibited substrate uptake by 75% (Fig. 5A and B). Long pretreatments with CCCP for up to 6 h should interfere with metabolism by uncoupling mitochondria. Therefore, the uptakes of valine and sucrose in the range of relatively low concentrations involve an important active component fuelled by ATP allowing the nutrient translocation through the plasma membrane. This is supported by the observations that compounds hindering ATPases activities inhibited valine and sucrose uptakes respectively by 82% and 79% with DES and 40% and 48% with DCCD. Orthovanadate, a more specific inhibitor of the plasma membrane Hþ-ATPase, inhibited valine and sucrose uptake of 22% and 27%, respectively. By contrast, experiments made with NaNO3, an inhibitor of tonoplast ATPase, did not modify the nutrient uptake. Altogether, these results sustain the plasma membrane Hþ-ATPase involvement in valine and sucrose absorptions. However, using FC, a fungal toxin known to activate specifically the Hþ-ATPase, the absorption of sucrose was not significantly modified whereas that of valine was increased by 30% (Fig. 6A and B). These latter results indicate that mechanisms sustaining valine and sucrose uptake may present some differences. The experiments carried out on PMV purified from beet roots clearly demonstrated that capacity of nutrient transport exists really at the plasmalemma level (Table 3). The uptake of both substrates by ‘energized’ PMVs was inhibited by the protonophore CCCP and may be related to a
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Figure 4. Eadie–Hofstee plots of valine (A) and sucrose (B) uptake by beet root slices incubated in a medium buffered at pH 5.3. Substate concentrations were in the range 0.1–100 mmol l1. Each point is the mean of data obtained on 15 slices7SE.
Table 1. Kinetic parameters of the three phases evidenced for valine and sucrose uptake by beet root tissues, calculated from the Eadie–Hofstee plots of Fig. 3 Substrate
Valine
Phases Km (mmol l1) Vm (pmol mg FW1 min1)
1 0.46 19.76
Sucrose 2 5.22 95.34
3 174.41 544.18
1 0.35 1.45
2 18.77 28.54
3 57.86 50.54
Table 2. Effect of 10 mmol l1 CCCP applied for 1 h on the uptake of valine and sucrose by beet root slices as a function of substrate concentration Substrate concentration (mmol l1)
0.5 1 10 100
Valine (pmol mgFW1)
Sucrose (pmol mg FW1)
Control
CCCP
I (%)
Control
CCCP
I (%)
180.0711.0 259.8718.0 707.6742.7 2716.67198.8
30.671.8 155.879.0 389.1722.4 1847.07185.8
83 60 45 32
37.373.6 49.076.2 179.4719.7 739.37188.8
7.170.9 26.875.0 89.5712.3 458.4792.2
81 55 50 38
Absorption duration was 30 min; results are means7SE (n ¼ 30 from two experiments).
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Figure 5. Effect of 10 mmol l1 CCCP pre-treatments lasting for durations up to 6 h on the uptake by beet root tissues of 1 mmol l1 valine (A) and 1 mmol l1 sucrose (B). Absorption duration : 30 min; control (active and passive absorption: black); CCCP (passive absorption: white); data are means7SE (n ¼ 30) from two independent experiments.
carrier activity with a broad range of specificity for amino acids since L-leucine inhibited strongly valine absorption, but more specific in the case of sucrose since glucose only showed a feeble competitive effect.
(Fig. 6C and D). These results suggest that the thiol and histidyl groups of protein carriers are involved in the uptake of valine and sucrose. These data are confirmed at the membrane level since PCMBS inhibited valine and sucrose uptake in ‘energized’ vesicles at a level similar to that observed in vivo (Table 3).
Involvement of thiols and histidyl groups on valine and sucrose uptake The effects of several products able to react specifically with thiol or histidyl groups were observed on sucrose and valine absorption. PCMBS and mersalyl are nonpermeant thiol reagents, whereas NEM is a permeant thiol reagent. DEPC is reactive with histidyl groups. Valine and sucrose uptakes were sensitive to these compounds, the percentage of inhibition being, respectively, 60% and 65% using PCMBS, 70% and 62% with mersalyl, 90% and 70% using NEM, 50% and 30% using DEPC
Relative involvement of transmembrane potential on valine and sucrose uptakes Addition of the discs induced continuous acidification of their incubation medium for 3 h as a result of the plasma membrane Hþ-ATPase activity (100–130 neq Hþ/h in average). The pH decreased in these experimental conditions from 6.3 to 5.5 and then remained stable at this value for several hours. FC, by activating this basal activity, induced a rapid decrease of the pH value to 4.9 in 2 h
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Figure 6. Comparative effects on the uptake of 1 mmol l1 valine (A,C) and 1 mmol l1 sucrose (B,D) by beet root slices pre-treated for 3 h with ATPase modulators (0.25 mmol l1 vanadate; 0.10 mmol l1 DES; 0.10 mmol l1 DCCD; 5 mmol l1 FC; 0.5 mol l1 NaNO3) and thiols reagents (NEM, PCMBS, mersalyl acid and DEPC at 1 mmol l1) Absorption duration : 30 min; C: control; data are means7SE (n ¼ 30) from two independent experiments.
Table 3. Effects of inhibitors or competing substrates on the amount of 1 mmol l1 valine and 1 mmol l1 sucrose recovered in the ‘energized’ PMV purified from beet roots at the end of a 2 min incubation period Substrate treatment
Valine (pmol mg prot1)
I (%)
Sucrose (pmol mg prot1)
I (%)
Control CCCP (10 mmol l1) PCMBS (1 mmol l1) Leucine (10 mmol l1) Glucose (10 mmol l1)
11.6871.07 4.8870.47 5.2970.50 5.3770.58 F
0 58 55 54
10.8870.97 4.0170.50 5.4670.45 F 9.0571.80
0 63 50 17
Data are means7SE (n ¼ 14 from two independent experiments).
(Fig. 7). This increased acidification thus indicated a large efflux of Hþ in the medium identical to that noted in other plant tissues. This efflux is expected to result in a membrane hyperpolarization as evidenced by electrophysiological data on various cells (Rubinstein and Cleland, 1981; Roblin et al., 1993). Following this FC treatment, as seen previously, valine uptake was increased whereas sucrose uptake was not significantly modified. In contrast, when external concentration of KCl was increased, valine uptake was strongly inhib-
ited, whereas sucrose uptake was activated even at very high KCl concentrations up to 100 mmol l1. At these high concentrations, KCl is known to depolarize the cell membrane (Komor et al., 1977; Etherton and Rubinstein, 1978). We have demonstrated this fact in our material by showing the increasing inhibition in [3H] TPPþ absorption in the presence of increasing amounts of KCl (Table 4). TPPþ has been used in various experiments as a probe to estimate the transmembrane potential gradient in various cell models (Lin, 1985, Komor
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Figure 7. (A) Representative effects of 10 mmol l1 valine, 10 mmol l1 sucrose and 10 mmol l1 FC on the time course of pH variations in the bathing medium of beet root slices; compounds added at arrow. (B) Corresponding amounts of Hþ excreted in the bathing medium as a function of time after application of valine and sucrose to the tissues; data are means7SD (n ¼ 7).
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Table 4. Effect of KCl at 1, 10 and 100 mmol l1 applied for various durations (1, 3, 6 h) on the uptake of 1 mmol l1 valine, 1 mmol l1 sucrose and 4 mmol l1 TPPþ by beet root tissues Time (h) KCl (mmol l1) Valine (pmol mg FW1) I (%) Sucrose (pmol mg FW1) I (%) TPPþ (pmol mg FW1) I (%) 1
0 1 10 100
10073 11875 10473 7172
F þ18 þ4 23
10.070.3 11.570.7 13.270.6 14.170.8
F þ15 þ32 þ41
F F F F
3
0 1 10 100
25078 24876 21676 16676
F 1 14 34
50.273.0 48.872.5 62.572.5 66.873.7
F 4 þ24 þ32
2.1970.05 2.0070.03 1.5370.02 0.9470.02
6
0 1 10 100
480714 497719 475712 280711
F þ4 22 42
70.073.0 73.174.6 80.775.6 84.973.7
F þ4 þ14 þ20
F 9 27 57
Uptake lasted for 30 min; data are means7SE (n ¼ 30 from two independent experiments).
and Tanner, 1976). TPPþ uptake at 100 mmol l1 KCl was reduced by 57%, which means a reduction of membrane potential by 25 mV. This value is probably underestimated since a part of the compound binds to anionic sites on cell walls. However, our results can be compared with those obtained by Rubinstein (1978) in oat leaf protoplasts. Thus, it was observed that KCl from 1 to 100 mmol l1 inhibited phosphonium salt uptake, respectively, by 27% and 65%. By comparison, a 60 mV depolarization was seen in oat cell coleoptiles with microelectrode methods when the KCl concentration was increased from 1 to 25 mmol l1. This experiment was validated by assay with FC, a fungal toxin known to hyperpolarize plant cell membrane. Applied at 10 mmol l1, FC increased TPPþ uptake by 27% (2.8270.09 vs. 2.227 0.06 pmol mg FW1 in control; n ¼ 40 from two independent experiments). Taken together, these data suggest that membrane potential differences may be the main driving component for valine uptake, whereas it appears less important in the case of sucrose uptake.
Relative involvement of Hþ and Kþ in valine and sucrose uptakes Addition of sucrose to the tissues induced a very weak and transient modification of the pH in the incubation medium (DpH of 0.09 units, maximum in 30 min). By contrast, addition of valine led to a pH increase of 0.35 units, lasting up to 5 h (Fig. 7A). Such proton influx has been linked to a Hþ/ substrate co-transport mechanism and, by comparison, the results obtained here suggest that valine and sucrose uptakes occurred according to differ-
ent characteristics in the tap root of sugar beet. Titration of protons excreted in the bathing medium corroborated the weak and transient mobilization of Hþ during sucrose uptake compared to valine co-transport processes (Fig. 7B). Following this observation that sucrose absorption may not be exclusively dependent on the Hþ mobilization, we examined another line of research implying the involvement of a possible Kþ regulation. This line was considered in the light of the data obtained on KCl effects (Table 4) and in accordance with the Kþ-substrate co-transport mechanism presented by Van Bel and Van Erven (1979) in tomato internode tissues. The results in Fig. 8 show that modulators of Kþ mobilization, acting in different ways, affected sucrose uptake more strongly than valine uptake. Thus, LiCl known to antagonize Kþ ions inhibited valine and sucrose uptake by 18% and 35%, respectively. TEA, which acts on Kþ conductance, doubled inhibition of sucrose uptake compared with valine uptake. When Kþ availability was suppressed from the incubation medium, either by using choline chloride or Nmethyl-D-glucamine, significant sucrose inhibition of uptake was observed (respectively, 30% and 37%) whereas valine absorption was not significantly modified. It is therefore suggested that, unlike the case of valine uptake, Kþ may play a major role in sucrose uptake.
Discussion Numerous data concerning nutrient uptake have been obtained using leaf tissues, probably for
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Figure 8. Effect of modulators of Kþ mobilization (TEA, LiCl, choline chloride and N-methyl D-glucamine applied at 10 mmol l1) on the uptake of 1 mmol l1 valine (A) and 1 mmol l1 sucrose (B) by beet root slices. Pre-treatment lasted for 3 h and absorption for 30 min; data are means7SE (n ¼ 45) from three independent experiments.
experimental convenience. By contrast, the transport of nutrients in the tap root has been less studied, although this storage organ is clearly more appropriate than the leaf (source of photoassimilates) for the study of nutrient accumulation. In the tap root, sucrose is mainly accumulated at the base and in the middle part of the organ. Nutrients are mainly stored in pericyclic cells (Saftner et al., 1983). In these cells, sucrose is stored in the vacuole and can be released from this compartment only after hydrolysis (Leigh, 1984). In mature sugar beet root, sucrose unloading is apoplastic and uptake and accumulation in the vacuole occur without hydrolysis (Wyse, 1979). The data presented here demonstrate the ability of the storage roots of sugar beet to take up sucrose and amino acids from the apoplast. The contribution of this process to metabolite transport from sieve tubes to the parenchyma cells is yet to be determined, but by considering the root structure,
the possibility of unloading from sieve tubes into the apoplast still exists.
Amino acids in the economy of the taproot In the past, research has been mainly focused on the transport and accumulation of sucrose in these sink organs. In this respect, amino acids have been ignored. However, sugar beet roots contain many Ncompounds, mainly proteins, betaine and amino acids (Schiweck et al., 1993). In particular, amino acids have been found in the phloem sap obtained by exudation (Fife et al., 1962) and after collection by the aphid stylet technique (Lohaus et al., 1994). Until now, little has been known about the process governing the accumulation of amino acids in beet root storage cells. Therefore, this study focused on the uptake mechanism of valine; however, a comparative study on sucrose absorption was made
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in light of what had already been carried out by certain others, but changing somewhat their experimental conditions. Wyse (1979), Saftner and Wyse (1980), Saftner et al. (1983) have studied sucrose absorption at high external sucrose concentrations (40–100 mmol l1) and for long incubation times (3–8 h). Our experiments were generally conducted at a concentration of 1 mmol l1 and incubation time was generally restricted to 30 min. This last parameter was chosen because Saftner et al. (1983) have shown that sucrose uptake takes place primarily into a compartment having a t12 of 21 min, presumably the cytoplasm, whereas uptake in vacuole shows a longer turnover time. Concerning the experimental concentration, we chose the 1 mmol l1 value because Saftner et al. (1983) have shown that this value is in the range of the active component demonstrated for low concentrations (below 20 mmol l1) and also because it is near that of valine content estimated in the root exudate (Lohaus et al., 1994). This also raises the question of the actual substrate concentration in the apoplast in particular at the contact of the wall surrounding the storage cells after unloading from the phloem. It is not proven that the very high sucrose concentration measured in the sieve tube was found as such at the interface, so that a much lower concentration can be available for the cell uptake. In this direction, it has been estimated that sucrose concentration in the apoplast of broadbean leaf should vary between 1 and 4.5 mmol l1 (Delrot et al., 1983). These conditions at a lower concentration of sucrose can also be encountered in some deleterious environmental conditions in which the formation and translocation of sucrose can be hindered. This was observed following treatment with Cd, a common pollutant from various industrial productions (Greger and Lindberg, 1986), and in the presence of air pollutants O3 and SO2 (Okano et al., 1984). Variations in climatic conditions also affect nutrient partitioning as shown in chilling stress (Giaquinta and Geiger, 1973) and to a lesser extent with water stress (Sung and Krieg, 1979). In these conditions, the passive phase must become less preponderant and the increasing activity of a carrier must overcome this effect.
Similarities in the transport characteristics of valine and sucrose First, it has been shown that aging was also operating in these storage tissues and induced the same uptake modifications for the two substrates. These data indicate that, in this context, valine and
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sucrose uptake share identical properties. In particular, by considering the results of Marvier et al. (1997) on red beet storage tissue, it is suggested that enhancement of valine transport may be due to an increased plasma membrane HþATPase activity rather than to changes in carrier activity or to changes in metabolism. Second, our results specify the kinetics parameters calculated for valine uptake and our calculations enable them to be compared with those of Saftner et al. (1983). The uptake in both cases took place according to 3 phases as a function of the concentration. Two phases, from 0.1 to 1 mmol l1 and from 1 to 10 mmol l1, respectively, showed properties of high affinity, corresponding to the active component of transport, whereas for higher concentration values up to 100 mmol l1, the nonsaturating uptake is normally indicative of a diffusion process. This was corroborated by the observation that treatment with CCCP was not completely efficient at these high nutrient concentrations. Third, in contrast to this last assumption, valine and sucrose uptakes in the range of millimolar concentrations were strongly dependent on an active process as shown by CCCP action. In fact, CCCP is a metabolic uncoupler that increases the apparent proton conductance and reduces the electrochemical potential of Hþ across membranes. Valine and sucrose uptakes were very sensitive to treatment with CCCP inducing a reduction up to 75% upon application of the inhibitor. This high value underlines the sensitivity of nutrient transport in sink cells to variations of metabolism. Alkali cation-stimulated ATPase activity has been found in sugar beet root tissues (Lindberg, 1980; Briskin and Poole, 1983). Alkali cations stimulated active sucrose uptake considerably while ionophores, which act on electrochemical potentials of Hþ, Kþ and Naþ across membranes, strongly inhibited transport (Saftner and Wyse, 1980). The putative ATPases inhibitors DES and DCCD inhibited active valine and sucrose uptakes. Moreover, unlike Saftner et al. (1983), our results show that uptakes took place primarily at the plasma membrane since uptake inhibition was triggered following treatment with orthovanadate, whereas the process was not disturbed after treatment with nitrate known as a inhibitor of tonoplast ATPase activity (VATPases) (Chanson, 1993). This capacity of transport at the plasmalemma level was definitely demonstrated by the uptake of both substrates by PMV purified from beet root tissues. The use of PMV allows a direct study of the activity of the plasma membrane transport to be made without interference of the energizing primary process (Hþ
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pump ATPase) and without effect of metabolism. Under artificial pmf, both substrates are absorbed in PMV following an active mechanism since treatment with CCCP, that abolished this pmf, inhibited strongly this uptake. Fourth, sensitivity of tissues and PMV to PCMBS and mersalyl, nonpermeant sulfhydryl reagents, suggest that valine and sucrose influx involved membrane proteins with essential SH groups accessible to the medium.
Differences between the transport of valine and sucrose The comparative study carried out in our conditions has highlighted important differences in the way the two metabolites are absorbed. Firstly, it was observed that tap root tissues have the ability to absorb valine 4 times faster on a molar basis than they absorb sucrose. This result was expected since a similar observation was made in the leaf tissues (Sakr et al., 1993). A second point of importance comes from the observation on the long-term experiments: valine uptake became constant after 12 h absorption whereas sucrose was continuously absorbed, though at a reduced rate, after 20 h of uptake. This latter observation might be related to the reduced amount of sucrose available in the apoplast since, in similar experiments, Rosenkranz et al. (2001) have shown that induction of invertase activity takes place one day after wounding. A third interesting point is the shift noted in the optimum pH for uptake, which is more acidic for valine than for sucrose. However, this difference in optima between the two substrates is not surprising given the fact that they are transported by different transporters. Nevertheless, the sharp relation between sucrose uptake and pH in our experiment should be noted, differing fundamentally from the data of Wyse (1979) whose results were obtained at a 100 mmol l1 sucrose concentration. This result however resembles those of Saftner and Wyse (1980) who showed that the active sucrose uptake in the vacuole is optimum at pH 6.5 when the substrate concentration was lowered to 20 mmol l1 (i.e. in the active phase). Our work also suggests that the uptake of valine and sucrose are not sustained by the same driving force since membrane potential difference appears as the main component in valine uptake by contrast with sucrose uptake. This can be deduced from the observations that FC, which is known to induce membrane hyperpolarization, enhanced valine uptake (unlike sucrose absorption) and that high
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external KCl concentrations, resulting in membrane depolarization, hindered valine uptake but increased sucrose absorption. Such stimulation of sugar loading by a high external Kþ concentration has previously been described in Ricinus petioles (Malek and Baker, 1977) and in beet tap roots (Saftner and Wyse, 1980). Another major difference can be linked to the uptake process itself by considering the relative Hþ and Kþ mobilization following addition of the considered substrate. Thus, the large and longlasting Hþ influx following application of the amino acid to the tissues argues for an Hþ-substrate cotransport, whereas an Hþ involvement is more restricted in the case of sucrose uptake. By contrast, arguments can be put forward in favour of a major Kþ participation in the uptake of sucrose. Firstly, we observed that effectors of Kþ, either Liþ antagonizing Kþ ions movements or TEA, an inhibitor of Kþ channels, reduced sucrose uptake more greatly than valine uptake. This implies that, in the transport of sucrose, Kþ channels may be activated and have to be opened for charge exhange to maintain a constant electrical potential. Second, the starvation of external Kþ, achieved by the use of choline chloride and Nmethyl-D-glucamine, inhibited sucrose uptake to a greater extent. The fact that this effect was somewhat limited can be explained by the high amount of Kþ found in the apoplast, in particular in the pectinaceous cell wall (Saftner and Wyse, 1980). These data argue for the hypothesis that sucrose is co-transported in root-sink cells with Kþ (Saftner and Wyse, 1980) and can be included in a model in which Kþ ions are necessary for a ‘‘potassionated’’ uptake system presented by Van Bel and Van Erven (1979) to explain the loading of this nutrient in tomato internode discs.
References Bennet AB, O’Neill SD, Spanswick RM. Hþ-ATPase activity from storage tissue of Beta vulgaris. Plant Physiol 1984;74:538–44. Bouche ´-Pillon S, Fleurat-Lessard P, Serrano R, Bonnemain JL. Asymmetric distribution of the plasma-membrane Hþ-ATPase in embryos of Vicia faba L. with special reference to transfer cells. Planta 1994;193:392–7. Briskin DP, Poole RJ. Characterization of a Kþ-stimulated adenosine triphosphatase associated with the plasma membrane of red beet. Plant Physiol 1983;71:350–5. Briskin DP, Reynolds-Niesman I. Determination of Hþ-ATP stoichiometry for the plasma membrane Hþ-ATPase from red beet (Beta vulgaris L.) storage tissue. Plant Physiol 1991;95:242–50.
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Briskin DP, Thornley WR, Wyse RE. Membrane transport in isolated vesicles from sugar beet tap root. I. Isolation and characterization of energy-dependent Hþ-transporting vesicles. Plant Physiol 1985a;78:865–70. Briskin DP, Thornley WR, Wyse RE. Membrane transport in isolated vesicles from sugar beet tap root. II. Evidence for a sucrose/Hþ-antiport. Plant Physiol 1985b;78: 871–5. Bush DR. Proton-coupled sucrose transport in plasmalemma vesicles isolated from sugar beet (Beta vulgaris L. cv Great Western) leaves. Plant Physiol 1989;89:1318–23. Bush DR. Electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport. Active transport of proton and calcium in higher plant cells. Plant Physiol 1990;93:1590–6. Chanson A. Active transport of proton and calcium in higher plant cells. Plant Physiol Biochem 1993;31:943–55. Cheung YNS, Nobel PS. Amino acid uptake by pea leaf fragments, specificity, energy sources and mechanism. Plant Physiol 1973;52:633–7. Chin C, Lee M, Weinstein M. Some characteristics of the sucrose uptake system of excised tomato roots. Can J Bot 1981;59:1159–63. Daie J. Sucrose uptake in isolated phloem of celery is a single saturable transport system. Planta 1987;171: 474–82. Delrot S, Bonnemain JL. Mechanism and control of phloem transport. Physiol Ve ´g 1985;23:199–230. Delrot S, Despeghel JP, Bonnemain JL. Phloem loading in Vicia faba leaves: effect of n-ethylmaleimide and parachloromercuribenzenesulfonic acid on Hþ extrusion, Kþ and sucrose uptake. Planta 1980;149: 144–8. Delrot S, Faucher M, Bonnemain JL, Bonmort J. Nycthemeral changes in intracellular and apoplastic sugars in Vicia faba leaves. Physiol Ve ´g 1983;21:459–67. Despeghel JP, Delrot S. Energetics of amino acid uptake by Vicia faba leaf tissues. Plant Physiol 1983;71:1–7. Ding B, Parthasarathy MV, Niklas K, Turgeon R. A morphometric analysis of the phloem-unloading pathway in developing tobacco leaves. Planta 1988;176: 307–18. Etherton B, Rubinstein B. Evidence for amino acid cotransport in oat coleoptiles. Plant Physiol 1978;61:933–7. Fife JM, Price C, Fife DC. Some properties of phloem exudate collected from root of sugar beet. Plant Physiol 1962;37:791–2. Fisher DG, Evert RF. Studies on the leaf of Amaranthus retroflexus (Amaranthaceae); ultrastructure, plasmodesmatal frequency and solute concentration in relation to phloem loading. Planta 1982;155:377–87. Fischer WN, Andre ´ B, Rentsch D, Krolkiewicz S, Tegeder M, Breitkreuz K, Frommer WB. Amino acid transport in plants. Trends Plant Sci 1998;3:188–95. Gahrtz M, L Stolz L, Sauer N. A phloem-specific sucroseHþ symporter from Plantago major L. supports the model of apoplastic phloem loading. Plant J 1994;6: 17–23.
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Gaillard C, Lemoine R, Delrot S. Absorption of L-valine by plasma membrane purified from sugar beet (Beta vulgaris L.) leaves. C R Acad Sci Paris Se ´r. III 1990;311:51–6. Gallet O, Lemoine R, Larsson C. Delrot S The sucrose carrier of the plant plasma membrane. I. Differential affinity labeling. Biochim Biophys Acta 1989;978: 56–64. " Getz HP, Grosclaude J, Kurkdjian A, Lelievre F, Maretzki A, Guern J. Immunological evidence for the existence of a carrier protein for sucrose transport in tonoplast vesicles from red beet (Beta vulgaris L.) root storage tissue. Plant Physiol 1993;102:751–60. Giaquinta RT. Sucrose hydrolysis in relation to phloem translocation in Beta vulgaris. Plant Physiol 1977;60: 339–43. Giaquinta RT. Phloem loading of sucrose. Annu Rev Plant Physiol 1983;34:347–87. Giaquinta RT. Sucrose translocation and storage in the sugar beet. Plant Physiol 1979;63:828–32. Giaquinta RT, Geiger DR. Mechanism of inhibition of translocation by localized chilling. Plant Physiol 1973;51:372–7. Greger M, Lindberg S. Effects of Cd 2þ and EDTA on young sugar beets (Beta vulgaris). I. Cd2þ uptake and sugar accumulation. Physiol Plant 1986;66:69–74. Haeder HE, Beringer H. Distribution of 14C assimilates in the storage root of sugar beet plants after import from leaves of different age. Physiol Plant 1987;70:590–6. Harper JF, Surowy TK, Sussman MR. Molecular cloning and sequence of cDNA encoding the plasma membrane proton pump (Hþ-ATPase) of Arabidopsis thaliana. Proc Natl Acad Sci USA 1989;86:1234–8. Hutchings VM. Sucrose and proton cotransport in Ricinus cotyledons. I. Hþ influx associated with sucrose uptake. Planta 1978;138:229–35. Komor E, Tanner W. The determination of the membrane potential of Chlorella vulgaris. Evidence for electrogenic sugar transport. Eur J Biochem 1976;70: 197–204. Komor E, Rotter M, Tanner W. A proton-co-transport system in a higher plant; sucrose transport in Ricinus communis. Plant Sci Lett 1977;9:153–62. Leigh RA. The role of the vacuole in the accumulation and mobilization of sucrose. Plant Growth Regul 1984;2: 339–46. Lemoine R, Daie J, Wyse RE. Evidence for the presence of a sucrose carrier in immature sugar beet tap roots. Plant Physiol 1988;86:575–80. Lemoine R, Bourquin S, Delrot S. Active uptake of sucrose by plant PMV: determination of some important physical and energetical parameters. Physiol Plant 1991;82:377–84. Lin W. Energetics of sucrose transport into protoplasts from developing soybean cotyledons. Plant Physiol 1985;78:41–5. Lindberg S. Kinetic studies of a (NaþþKþþMg2þ) ATPase in sugar beet roots. III. A proposed model for the (NaþþKþ) activation and its significance for field properties. Plant Physiol 1980;48:65–70.
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Lohaus G, Burba M, Heldt HW. Comparison of the contents of sucrose and amino acids in the leaves, phloem sap and taproots of high and low sugarproducing hybrids of sugar beet (Beta vulgaris L.). J Exp Bot 1994;45:1097–101. Malek F, Baker DA. Proton co-transport of sugars in phloem loading. Planta 1977;135:297–9. Marvier AC, Williams LE, Leigh RA, Hall JL. Effects of prolonged washing on primary and secondary transport processes at the plasma membrane in red beet storage tissue. Plant Physiol 1997;115:263–72. Okano K, Ito O, Takeba G, Shimizu A, Totsuka T. Alteration of 13C-assimilate partitioning in plants of Phaseolus vulgaris exposed to ozone. New Phytol 1984;97:155–63. Oleski NA, Bennett AB. Hþ-ATPase activity from storage tissue of Beta vulgaris. IV. N,N0 -dicyclohexylcarbodiimide binding and inhibition of the plasma membrane Hþ-ATPase. Plant Physiol 1987;83:569–72. O’Neill SD, Spanswick RM. Effects of vanadate on the plasma membrane ATPase of red beet and corn. Plant Physiol 1984;75:586–91. Otsiogo-Oyabi H, Roblin G. Uptake of glycine by excised pulvini of Mimosa pudica in relation to proton pump activity. Physiol Plant 1985;65:40–4. Poole RJ, Briskin DP, Kratky Z, Johnstone RM. Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet. Plant Physiol 1984;74:549–56. Racusen RH, Galston AW. Electrical evidence for rhythmic changes in the cotransport of sucrose and hydrogen ions in Samanea pulvini. Planta 1977;135:57–62. Riesmeier JW, Willmitzer L, Frommer WB. Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast. EMBO J 1992;11:4705–13. Roblin G, Moyen C, Fleurat-Lessard P, Bonmort J. Effect of methylxanthines on seismonastic reaction, proton flux, membrane potential, respiration and calcium uptake in motor cells of Mimosa pudica pulvinus. J Plant Physiol 1993;142:197–202. Rosenkranz H, Vogel R, Greiner S, Rausch T. In wounded sugar beet (Beta vulgaris L.) tap-root, hexose accumulation correlates with the induction of a vacuolar invertase isoform. J Exp Bot 2001;52:2381–5. Rubinstein B. Use of lipophilic cations to measure the membrane potential of oat leaf protoplasts. Plant Physiol 1978;62:927–9. Rubinstein B, Cleland RE. Responses of Avena coleoptiles to suboptimal fusicoccin: kinetics and comparisons with indolacetic acid. Plant Physiol 1981;68:543–7. Saftner RA, Wyse RE. Alkali cation sucrose co-transport in the root sink of sugar beet. Plant Physiol 1980;66: 884–9.
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Saftner RA, Daie J, Wyse RE. Sucrose uptake and compartmentation in sugar beet taproot tissue. Plant Physiol 1983;72:1–6. Sakr S, Lemoine R, Gaillard C, Delrot S. Effect of cutting on solute uptake by plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves. Plant Physiol 1993;103:49–58. Sauer N, Stolz J. SUC1 and SUC2, two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker’s yeast and identification of the histidine-tagged protein. Plant J 1994;6:67–77. Schmalstig JG, Geiger DR. Phloem unloading in developing leaves of sugar beet. I. Evidence for pathway through the symplast. Plant Physiol 1985;79: 237–41. Schiweck H, Jeanteur-De-Beukelaer C, Vogel M. Das Verhalten der stickstoffhaltigen Nichtzuckerstoffe . von Ruben wa. hrend des Fabrikationsprozesses. Zuckerindustrie 1993;118:15–23. Secor J. Regulation of sucrose efflux from soybean leaf discs. Plant Physiol 1987;83:143–8. Serrano R. Structure and function of plasma membrane ATPase. Annu Rev Plant Physiol Plant Mol Biol 1989;40:61–94. Servaites J, Schrader LE, Jung DM. Energy-dependent loading of amino acids and sucrose into the phloem of soybean. Plant Physiol 1979;64:546–50. Snyder FW, Carlson GE. Photosynthate partitioning in sugar beet. Crop Sci 1978;18:657–69. Sovonick SA, Geiger DR, Fellows RJ. Evidence for active phloem loading in the minor veins of sugar beet. Plant Physiol 1974;54:886–91. Stein M, Willenbrink J. On accumulation of sucrose in the growing sugar beet. Z Pflanzenphysiol 1976;79: 310–22. Sung FJM, Krieg DR. Relative sensitivity of photosynthetic assimilation and translocation of 14carbon to water stress. Plant Physiol 1979;64:852–6. Sze H. Hþ-translocating ATPases: advances using membrane vesicles. Annu Rev Plant Physiol 1985;36: 175–208. Van Bel AJE, Kempers R. Symplastic isolation of the sieve element-companion cell complex in the phloem of Ricinus communis and Salix alba stems. Planta 1990;183:69–76. Van Bel AJE, Van Erven AJ. A model for proton and potassium co-transport during the uptake of glutamine and sucrose by tomato internode discs. Planta 1979;145:77–82. Wyse RE. Sucrose uptake by sugar beet tap root tissue. Plant Physiol 1979;84:1088–95. Wyse RE, Zamski E, Deri Tomos A. Turgor regulation of sucrose transport in sugar beet taproot tissue. Plant Physiol 1986;81:478–81.
www.elsevier.de/jplph
Valine uptake in the tap root of sugar beet: a comparative analysis with sucrose uptake Philippe Michonneau, Gabriel Roblin, Janine Bonmort, Pierrette Fleurat-Lessard* Laboratoire de Physiologie, Biochimie et Biologie mole´culaire Ve´ge´tales, Universite´ de Poitiers, UMR CNRS 6161, 40 Avenue du Recteur Pineau, 86022 Poitiers, Cedex, France Received 27 November 2003; accepted 23 February 2004
KEYWORDS Beta vulgaris; Valine uptake; Sucrose uptake; Nutrient storage; Tap root
Summary Given the lack of data on the absorption of amino acids in the tap root of Beta vulgaris, we studied the uptake of valine and compared it with that of sucrose at the same concentration (1 mM). The uptake of both substrates shared some similar characteristics. In particular, the absorption in both cases was controlled by an active process as evidenced by the inhibitory effect of CCCP and inhibitors of ATPases (DES, DCCD, orthovanadate). Both absorptions also involved the thiol and histidyl groups of protein carriers included in the plasmalemma as shown by treatment with specific compounds (PCMBS, mersalyl, NEM) inhibiting the transport of the nutrients in tissues and in purified PMV. However, it was shown that these uptakes present major differences. Firstly, unlike sucrose uptake, valine uptake was very sensitive to transmembrane electrical potential. Indeed, hyperpolarizing treatment with FC increased valine uptake but did not modify sucrose uptake. By contrast, treatment with high concentrations of KCl, which should result in depolarization of the cells, considerably decreased valine uptake and activated sucrose uptake. Secondly, ion mobilizations were different in the two types of transport. Unlike sucrose, application of valine to tissues strongly modified the time course of Hþ influx. By contrast, sucrose uptake was controlled by Kþ involvement as shown by effects either of modulators of Kþ mobilization (LiCl, TEA) or of treatments inducing Kþ starvation from the external medium. & 2004 Elsevier GmbH. All rights reserved.
Abbreviations: CCCP, Carbonyl cyanide m-chlorophenylhydrazone; DCCD, N,N0 -Dicyclohexylcarbodiimide; Dc, Membrane electrical potential; DEPC, Diethylpyrocarbonate; DES, Diethylstilbestrol; FC, Fusicoccin; HEPES, N–[2-hydroxyethyl]piperazine-N0 –[2-ethanesulfonic acid]; MES, 2-[N-morpholino]ethanesulfonic acid; NEM, N-ethylmaleimide; PCMBS, p-Chloromercuribenzenesulfonic acid; PMF, Proton motive force; PMV, Plasma membrane vesicles; TEA, Tetraethylammonium chloride; TPPþ, Tetraphenylphosphonium bromide *Corresponding author. Fax: þ33-5-49-45-41-86. E-mail address: [email protected] (P. Fleurat-Lessard). 0176-1617/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2004.02.005
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Introduction During plant development, photoassimilates are exchanged between sources, suppliers of metabolites (exporting leaves), and sinks, importers of metabolites (growing or storage organs). The long distance transport of photoassimilates is controlled by the osmotic gradient established in the phloem between the source and the sinks (Delrot and Bonnemain, 1985, and references therein). The first transport of assimilates occurs from the photosynthetic leaf cells into the leaf vascular system. Sucrose transport from mesophyll tissues to sites of phloem loading has been shown to be a regulated process and does not result from simple leakage (Secor, 1987). The route for transport is through the apoplast requiring active uptake by sieve tubes and contiguous cells (Giaquinta, 1983; Fisher and Evert, 1982). More specifically, the hypothesis is that sucrose absorption into the vascular tissues is mediated by an Hþ-sucrose transport (Giaquinta, 1983), a hypothesis sustained by the results of experiments with intact tissues (Daie, 1987; Gahrtz et al., 1994). In addition, physiological data have shown that phloem loading of amino acids and sucrose is mediated by different and separate carriers, all of which are dependent on an energy-requiring mechanism (Servaites et al., 1979; Sovonick et al., 1974). In contrast to phloem loading in mature leaves, few data deal with phloem unloading in sinks such as developing leaves (Schmalstig and Geiger, 1985) or storage roots (Giaquinta, 1979). As a general rule, mechanisms regulating the unloading of nutrients, from conducting to storage tissues, are not clearly understood; the pathway may be via plasmodesmatal connections in the symplast (Schmalstig and Geiger, 1985; Ding et al., 1988) or may be apoplastic (Van Bel and Kempers, 1990). There is evidence that sucrose translocated into sugar beet root is unloaded into the free space (Stein and Willenbrink, 1976) and is taken up in an unhydrolyzed form by storage parenchyma cells via a nonsaturating carrier, thereby arguing for an apoplastic pathway (Wyse, 1979). At the cell level, absorption of sucrose and amino acids has been studied on various experimental models, leaf fragments (Cheung and Nobel, 1973; Delrot et al., 1980; Despeghel and Delrot, 1983), coleoptiles (Etherton and Rubinstein, 1978), cotyledons (Komor et al., 1977; Hutchings, 1978), roots (Chin et al., 1981), and pulvinar tissues (Racusen and Galston, 1977; Otsiogo-Oyabi and Roblin, 1985). Whether these models are relevant is questionable since the cells in normal conditions in planta behave like source cells and are thus mainly
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concerned with unloading processes. However, in these particular experimental conditions, in which tissues were isolated from normal correlations encountered in planta, it has been shown that the energy of these transports may be provided by a gradient of electrochemical potential of protons across a membrane that could be generated and maintained by a vectorial membrane-bound ATPase. Advances towards a complete understanding of transport processes can be attributed to the use of isolated membrane vesicles confirming the above-defined data (Bush, 1989, 1990; Gaillard et al., 1990). Moreover, molecular approaches have shown that nutrient transport occurs by means of specific carriers (Riesmeier et al., 1992; Sauer and Stolz, 1994; Fischer et al., 1998). As a corollary, Hþ-ATPase pumps necessary to energize nutrient transport have been purified, characterized, localized, and cloned (Sze, 1985; Harper et al., 1989; Serrano, 1989; Bouche ´-Pillon et al., 1994). Our work was focused on the study of nutrient accumulation in a major sink organ, the tap root of Beta vulgaris L. As the transport of assimilates affects plant productivity, a better partitioning rather than a high photosynthesis rate will increase the sugar beet yield. The sugar beet is a biennial plant accumulating nutrients in its tap root during the first year and using this store in the second year to form reproductive organs. Following the distribution of sugars in tap root after assimilation of 14C by old and young leaves, Haeder and Beringer (1987) showed that more sucrose was found in the centre of the storage tissue in relation to the course of the vascular bundle extending from the source leaf. The tap root has an unusual structure, since conducting tissues are organized into concentric rings produced by successive supernumerary cambial layers. Nutrients are stored in xylem parenchyma and, to a greater extent, in the large and outermost pericyclic cells (i.e. storage cells in each vascular ring). In tap roots cultivated in our conditions, the determination of sucrose content reached 8176 mg g1 fresh weight (n ¼ 3). However, this average value obtained from root tissue homogenates certainly underestimates the actual sucrose concentration in the vacuole of storage cells. Previous results obtained on nutrient transport in the tap root of red and sugar beet were concerned essentially with sucrose transport in the vacuolar compartment. Based on tissue analysis, it has been shown that a major portion of the sucrose taken up from the free space is stored in the vacuole without hydrolysis (Giaquinta, 1977; Wyse, 1979). The downhill electrochemical potential gradient across
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the tonoplast is coupled with sucrose uptake via a proton antiport system and a Kþ gradient. The Hþantiport process was confirmed by use of tonoplast vesicles isolated from beet storage tissue (Briskin et al., 1985b) in which an Hþ-ATPase sensitive to nitrate was isolated (Briskin et al., 1985a). Finally, the existence of a carrier protein involved in sucrose uptake in tonoplast was demonstrated by an immunological approach (Getz et al., 1993). The question of sucrose transport at the plasmalemma has only been reported in immature sugar beet tap roots according to a classical sucrose Hþcotransport mechanism (Lemoine et al., 1988) but no true demonstration has been afforded and investigations along these lines have not been further pursued; however, this mechanism can be expected since an Hþ-ATPase activity has been demonstrated in the plasma membrane isolated from tap root tissues in red beet (O’Neill and Spanswick, 1984; Bennett et al., 1984; Poole et al., 1984; Oleski and Bennett, 1987; Briskin and Reynolds-Niesman, 1991) and in sugar beet in the course of this work. In any event, data related to the sucrose uptake at the plasmalemma level in the tap root of the sugar beet are scarce, and characteristics of amino acid uptake are completely unknown. However, many N-compounds and amino acids in particular have been detected in this organ (Schiweck et al., 1993). It was further shown that glutamate, glutamine and aspartate are the main amino acids transported from the leaf to the root in sugar beet. However, many neutral amino acids are translocated and, among these, valine is found in the phloem sap at a concentration that is estimated to be about 3 mmol l1 (Lohaus et al., 1994). Valine was chosen in the present study because the uptake mechanism of neutral amino acids has been well characterized in many plant materials and, more precisely, the uptake of valine by beet leaf cells has been widely investigated in our laboratory, thus allowing a direct comparison with the results obtained here with those made on root cells. In this context, the main features of our work were to analyse the uptake of valine, used at a relatively low concentration (1 mmol l1), and to compare it with the uptake of sucrose, also used at the same experimental conditions. In this regard, we stress that previously, except in experiments concerning the determination of kinetic parameters of sucrose unloading (Wyse et al., 1986), high concentrations (5–100 mmol l1) of substrates were employed to study uptake mechanisms in the tap root of B. vulgaris. In the present work, we first characterized the absorption of valine by beet root tissues by
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determining uptake pattern, energetics of the process, and implication of a protein component. The data thus obtained were compared throughout this work with that observed on sucrose uptake. Secondly, using PMV, we demonstrated that valine and sucrose uptake in the tap root of B. vulgaris occurred initially at the plasmalemma level in the sink cells. Thirdly, we evidenced features differentiating valine from sucrose uptake. In particular, unlike valine uptake that shares the general properties of an Hþ-substrate co-transport, sucrose uptake was shown to be poorly related to Hþ influx and transmembrane potential but correlated to a Kþ mobilization in a way needing further investigations to be more precise.
Materials and methods Plant material Sugar beets (B. vulgaris var. aramis) were grown in a greenhouse on vermiculite under natural conditions. The plants were watered daily with Snyder and Carlson (1978) nutritive solution. Supplementary lightning was provided with Osram 58/10 fluorescent tubes, giving 30 mmol photon m2 s1 from 6.00 to 9.00 a.m. and from 7.00 to 10.00 p.m. Plants were used for experiments when they were about 2 months old and bearing ten expanded leaves.
Transport assays in vivo Responses on intact systems are complicated by several factors, including metabolic fates due to aging, wounding, and compartmentalization of the substrates. These constraints were tentatively restricted through careful experimental conditions: sampling was made in a precise zone of the root, and tissue handling duration was reduced. Tissue cylinders of 12 mm diameter were taken from the root cortex with a cork borer. These tissues were sliced with a razor blade into discs 1 mm in thickness and 0.110070.0013 g in mass. These discs were divided into four quarters of 0.026870.0030 g, to facilitate uptake of the substrates. Unless stated otherwise, the quarters were immersed for 1 h in a solution at pH 5.30, containing 20 mmol l1 MES/NaOH, 0.25 mmol l1 MgCl2, 0.5 mmol l1 CaCl2, 300 mmol l1 sorbitol (added to maintain osmotic pressure in the root cells, which have been isolated from their natural environment as a result of disc sampling). The tissues were then transferred in the same medium containing
ARTICLE IN PRESS 1302 1 mmol l1 [3H]valine (final activity 9.25 kBq ml1) and 1 mmol l1 [14C] sucrose (final activity 11 kBq ml1). Incubation (generally 30 min) was run under mild agitation on a shaker at 251C. Experiments were run in parallel on both substrates on the same set of discs to compare accurately and quantitatively the modifications noted following various treatments. At the end of the incubation, the quarters were rinsed (3 3 min) in the preincubation solution to remove the apoplastic label and were placed in scintillation vials. Digestion occurred at 551C during 24 h. in a mixture of perchloric acid (56%), H2O2 (27%), and 0.1% Triton 100 (17%). Finally, 4 ml of scintillation liquid (Ecolite TM, ICN) was added to the vials, and radioactivity was counted by liquid scintillation spectroscopy (1900 TR Packard). In order to estimate the transmembrane potential gradient in beet root tissues, TPPþ was used as probe. The beet root slices were incubated and treated exactly as described above, except that assimilates in the incubation medium were replaced with 4 mmol l1 [3H] TPPþ (final activity: 2.17 kBq ml1) (Amersham, France).
Transport assays in vitro Purified PMV were prepared according to the general method of Gallet et al. (1989) from the beet roots and used to measure uptake of valine and sucrose in various energizing conditions (Lemoine et al., 1991). Briefly, the PMV at a concentration of 15 mg protein ml1 were equilibrated in a medium containing 0.3 mol l1 sorbitol, 50 mmol l1 potassium phosphate (pH 7.5), 0.5 mmol l1 CaCl2, 0.25 mmol l1 MgCl2 and 0.5 mmol l1 DTT. The PMV were then resuspended in 50 mmol l1 sodium phosphate at pH 5.5 containing 10 mmol l1 valinomycin (condition creating DpH and DC gradients: PMV in ‘energized’ state). Valinomycin creates a DC gradient following the diffusion of Kþ outwards the vesicles in the presence of the K ionophore. In every case, 1 mmol l1 [3H] valine (final activity: 41 kBq ml1) and 1 mmol l1 [U-14C]sucrose (27 kBq ml1) were added in the medium. Uptake was initiated by rapidly mixing vesicles with the incubation medium on a vortex apparatus. During experiments, solutions and test tubes were kept in a water bath maintained at 251C. At the desired time, uptake was terminated by adding 2 ml of the used medium plus 5 mmol l1 HgCl2 and filtering the content of the test tube on a Millipore HAWP filter (pore size 0.45 mm) prewetted with 2 ml rinsing medium. The test tube was rinsed with 2 ml of medium and
P. Michonneau et al.
poured onto the filter. The filters were then placed in a scintillation vial, dried at 501C for 1 h and immersed in 4 ml of scintillation liquid for counting.
Hþ Fluxes measurements Tap root tissues prepared as described above were preincubated for 1 h in 15 ml of a medium containing 300 mmol l1 sorbitol, 0.25 mmol l1 MgCl2, and 0.5 mmol l1 CaCl2. The quarters were then transferred to 10 ml fresh medium. Variation of the pH of the incubation medium were read on a pH-meter (Expandomatic SS2 Beckman, Roissy, Fr) provided with a combined electrode (Futura, Beckman) and linked to a potentiometric recorder. The incubation medium was aerated with a rod stirrer (Metrohm). Substrates were added in the medium as indicated in the Fig. 7A. In a series of experiments, Hþ amount was titrated either by 5 103 N HCl or 5 103 N NaOH in 2 ml aliquot withdrawn at regular intervals (generally 30 min) from the incubation medium. This experimental value was expressed for the whole incubation volume and by taking into account the weight of fresh root tissues.
Results Characteristics of valine and sucrose uptake Tap root tissues took up labelled valine and sucrose from a 1 mmol l1 solution following three successive linear components as a function of uptake duration. The general shape of absorption did not show differences between the two substrates except that valine was absorbed in a much higher amount on a molar basis than was sucrose. In both cases, the first phase, lasting 3 h, was followed by a second phase, lasting between 3 and 12 h, which was characterized by a steeper slope. The third phase, after 12 h, differed somewhat between the two substrates. In this last step, valine uptake rate became stationary whereas sucrose uptake remained high up to 36 h (Fig. 1). This long-term uptake study is however difficult to interpret because of metabolism and compartmentation of the transported molecules. In this regard, the data suggested that valine uptake may be inhibited by internal valine content or counterbalanced by an efflux, whereas sucrose uptake may continue due to its metabolization. As a practical indication, our results indicated that a 30 min-absorption time was convenient for further studies concerning the mechanism of nutrient uptake.
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Figure 1. Typical time course of 1 mmol l1 valine and 1 mmol l1 sucrose uptake by beet root slices at pH 5.3; data are means7SE (n ¼ 15). Inset: data in the first 2 h. Experiment has been made 3 times with similar results.
Figure 2. Effect of ageing in root tissues of sugar beet on the uptake of 1 mmol l1 valine and 1 mmol l1 sucrose at pH 5.3. Uptake duration: 30 min; data are means7SE (n ¼ 30) from two independent experiments.
In the course of long-term uptake, it has been previously shown that the aging of sugar beet leaf discs considerably promotes the uptake of sugars and amino acids (Sakr et al., 1993). Aging is achieved by floating the root quarters for several hours on the incubation medium before transferring the tissues to the uptake medium containing the labelled substrates. The curves in Fig. 2 show that only a small modification in the capacity of valine and sucrose absorption was seen during the first 3 h. After this lag, uptake of both substrates increased continuously and considerably until 12 h of aging. After this period, a significant
decrease was noted. Therefore, in subsequent experiments, uptake was carried out 3 h after root tissue sampling. The uptake of both valine and sucrose were shown to be dependent on the experimental pH (Fig. 3). A clear difference appears between the two substrates since it was observed that absorption was optimal at pH 5.2 for valine and pH 6.0 for sucrose. In light of these results, further experiments were carried out using MES buffer adjusted to pH 5.3 since at this value absorption of both substrates was close to the optimum and processed near the apoplastic pH.
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Figure 3. Effect of pH and buffer on the uptake of 1 mmol l1 valine and 1 mmol l1 sucrose by beet root slices. Root tissues were preincubated 3 h in buffered medium at indicated pH and uptake lasted 30 min. Two different buffers were used: 20 mmol l1 HEPES buffer for pH 4.0, 5.2 and 8.0; 20 mmol l1 MES buffer for pH 6.0 and 7.0. Data are means7SE (n ¼ 30) from two independent experiments.
Kinetic parameters of valine and sucrose uptake Concentration dependence studies were run with valine and sucrose concentrations ranging from 0.1 to 100 mmol l1. The data enabled a saturable component up to 10 mmol l1 to be distinguished (suggesting that part of assimilate uptake is carrier mediated) and a more or less linear component at higher concentrations. Eadie–Hofstee plots (Fig. 4) illustrate these data and specify that the saturable phase was composed of two components characterized by different apparent kinetic parameters shown in Table 1.
Characterization of an active process The relative active and passive components of valine and sucrose absorptions have been characterized by the use of the protonophore CCCP. Treatment with 10 mmol l1 CCCP for 1 h showed that the passive components of valine and sucrose absorptions increased when the nutrient concentration increased (near 15–20% at 0.5 mmol l1 but 60–70% at 100 mmol l1) (Table 2). Increasing the length of the CCCP treatment (Fig. 5A and B) showed a constant residual uptake of the substrates applied at 1 mmol l1. This indicated that a weak passive component occurred both for valine and sucrose absorption, which remained constant throughout the course of the experiment. For
example, a 6 h treatment inhibited substrate uptake by 75% (Fig. 5A and B). Long pretreatments with CCCP for up to 6 h should interfere with metabolism by uncoupling mitochondria. Therefore, the uptakes of valine and sucrose in the range of relatively low concentrations involve an important active component fuelled by ATP allowing the nutrient translocation through the plasma membrane. This is supported by the observations that compounds hindering ATPases activities inhibited valine and sucrose uptakes respectively by 82% and 79% with DES and 40% and 48% with DCCD. Orthovanadate, a more specific inhibitor of the plasma membrane Hþ-ATPase, inhibited valine and sucrose uptake of 22% and 27%, respectively. By contrast, experiments made with NaNO3, an inhibitor of tonoplast ATPase, did not modify the nutrient uptake. Altogether, these results sustain the plasma membrane Hþ-ATPase involvement in valine and sucrose absorptions. However, using FC, a fungal toxin known to activate specifically the Hþ-ATPase, the absorption of sucrose was not significantly modified whereas that of valine was increased by 30% (Fig. 6A and B). These latter results indicate that mechanisms sustaining valine and sucrose uptake may present some differences. The experiments carried out on PMV purified from beet roots clearly demonstrated that capacity of nutrient transport exists really at the plasmalemma level (Table 3). The uptake of both substrates by ‘energized’ PMVs was inhibited by the protonophore CCCP and may be related to a
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Figure 4. Eadie–Hofstee plots of valine (A) and sucrose (B) uptake by beet root slices incubated in a medium buffered at pH 5.3. Substate concentrations were in the range 0.1–100 mmol l1. Each point is the mean of data obtained on 15 slices7SE.
Table 1. Kinetic parameters of the three phases evidenced for valine and sucrose uptake by beet root tissues, calculated from the Eadie–Hofstee plots of Fig. 3 Substrate
Valine
Phases Km (mmol l1) Vm (pmol mg FW1 min1)
1 0.46 19.76
Sucrose 2 5.22 95.34
3 174.41 544.18
1 0.35 1.45
2 18.77 28.54
3 57.86 50.54
Table 2. Effect of 10 mmol l1 CCCP applied for 1 h on the uptake of valine and sucrose by beet root slices as a function of substrate concentration Substrate concentration (mmol l1)
0.5 1 10 100
Valine (pmol mgFW1)
Sucrose (pmol mg FW1)
Control
CCCP
I (%)
Control
CCCP
I (%)
180.0711.0 259.8718.0 707.6742.7 2716.67198.8
30.671.8 155.879.0 389.1722.4 1847.07185.8
83 60 45 32
37.373.6 49.076.2 179.4719.7 739.37188.8
7.170.9 26.875.0 89.5712.3 458.4792.2
81 55 50 38
Absorption duration was 30 min; results are means7SE (n ¼ 30 from two experiments).
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Figure 5. Effect of 10 mmol l1 CCCP pre-treatments lasting for durations up to 6 h on the uptake by beet root tissues of 1 mmol l1 valine (A) and 1 mmol l1 sucrose (B). Absorption duration : 30 min; control (active and passive absorption: black); CCCP (passive absorption: white); data are means7SE (n ¼ 30) from two independent experiments.
carrier activity with a broad range of specificity for amino acids since L-leucine inhibited strongly valine absorption, but more specific in the case of sucrose since glucose only showed a feeble competitive effect.
(Fig. 6C and D). These results suggest that the thiol and histidyl groups of protein carriers are involved in the uptake of valine and sucrose. These data are confirmed at the membrane level since PCMBS inhibited valine and sucrose uptake in ‘energized’ vesicles at a level similar to that observed in vivo (Table 3).
Involvement of thiols and histidyl groups on valine and sucrose uptake The effects of several products able to react specifically with thiol or histidyl groups were observed on sucrose and valine absorption. PCMBS and mersalyl are nonpermeant thiol reagents, whereas NEM is a permeant thiol reagent. DEPC is reactive with histidyl groups. Valine and sucrose uptakes were sensitive to these compounds, the percentage of inhibition being, respectively, 60% and 65% using PCMBS, 70% and 62% with mersalyl, 90% and 70% using NEM, 50% and 30% using DEPC
Relative involvement of transmembrane potential on valine and sucrose uptakes Addition of the discs induced continuous acidification of their incubation medium for 3 h as a result of the plasma membrane Hþ-ATPase activity (100–130 neq Hþ/h in average). The pH decreased in these experimental conditions from 6.3 to 5.5 and then remained stable at this value for several hours. FC, by activating this basal activity, induced a rapid decrease of the pH value to 4.9 in 2 h
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Figure 6. Comparative effects on the uptake of 1 mmol l1 valine (A,C) and 1 mmol l1 sucrose (B,D) by beet root slices pre-treated for 3 h with ATPase modulators (0.25 mmol l1 vanadate; 0.10 mmol l1 DES; 0.10 mmol l1 DCCD; 5 mmol l1 FC; 0.5 mol l1 NaNO3) and thiols reagents (NEM, PCMBS, mersalyl acid and DEPC at 1 mmol l1) Absorption duration : 30 min; C: control; data are means7SE (n ¼ 30) from two independent experiments.
Table 3. Effects of inhibitors or competing substrates on the amount of 1 mmol l1 valine and 1 mmol l1 sucrose recovered in the ‘energized’ PMV purified from beet roots at the end of a 2 min incubation period Substrate treatment
Valine (pmol mg prot1)
I (%)
Sucrose (pmol mg prot1)
I (%)
Control CCCP (10 mmol l1) PCMBS (1 mmol l1) Leucine (10 mmol l1) Glucose (10 mmol l1)
11.6871.07 4.8870.47 5.2970.50 5.3770.58 F
0 58 55 54
10.8870.97 4.0170.50 5.4670.45 F 9.0571.80
0 63 50 17
Data are means7SE (n ¼ 14 from two independent experiments).
(Fig. 7). This increased acidification thus indicated a large efflux of Hþ in the medium identical to that noted in other plant tissues. This efflux is expected to result in a membrane hyperpolarization as evidenced by electrophysiological data on various cells (Rubinstein and Cleland, 1981; Roblin et al., 1993). Following this FC treatment, as seen previously, valine uptake was increased whereas sucrose uptake was not significantly modified. In contrast, when external concentration of KCl was increased, valine uptake was strongly inhib-
ited, whereas sucrose uptake was activated even at very high KCl concentrations up to 100 mmol l1. At these high concentrations, KCl is known to depolarize the cell membrane (Komor et al., 1977; Etherton and Rubinstein, 1978). We have demonstrated this fact in our material by showing the increasing inhibition in [3H] TPPþ absorption in the presence of increasing amounts of KCl (Table 4). TPPþ has been used in various experiments as a probe to estimate the transmembrane potential gradient in various cell models (Lin, 1985, Komor
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Figure 7. (A) Representative effects of 10 mmol l1 valine, 10 mmol l1 sucrose and 10 mmol l1 FC on the time course of pH variations in the bathing medium of beet root slices; compounds added at arrow. (B) Corresponding amounts of Hþ excreted in the bathing medium as a function of time after application of valine and sucrose to the tissues; data are means7SD (n ¼ 7).
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Table 4. Effect of KCl at 1, 10 and 100 mmol l1 applied for various durations (1, 3, 6 h) on the uptake of 1 mmol l1 valine, 1 mmol l1 sucrose and 4 mmol l1 TPPþ by beet root tissues Time (h) KCl (mmol l1) Valine (pmol mg FW1) I (%) Sucrose (pmol mg FW1) I (%) TPPþ (pmol mg FW1) I (%) 1
0 1 10 100
10073 11875 10473 7172
F þ18 þ4 23
10.070.3 11.570.7 13.270.6 14.170.8
F þ15 þ32 þ41
F F F F
3
0 1 10 100
25078 24876 21676 16676
F 1 14 34
50.273.0 48.872.5 62.572.5 66.873.7
F 4 þ24 þ32
2.1970.05 2.0070.03 1.5370.02 0.9470.02
6
0 1 10 100
480714 497719 475712 280711
F þ4 22 42
70.073.0 73.174.6 80.775.6 84.973.7
F þ4 þ14 þ20
F 9 27 57
Uptake lasted for 30 min; data are means7SE (n ¼ 30 from two independent experiments).
and Tanner, 1976). TPPþ uptake at 100 mmol l1 KCl was reduced by 57%, which means a reduction of membrane potential by 25 mV. This value is probably underestimated since a part of the compound binds to anionic sites on cell walls. However, our results can be compared with those obtained by Rubinstein (1978) in oat leaf protoplasts. Thus, it was observed that KCl from 1 to 100 mmol l1 inhibited phosphonium salt uptake, respectively, by 27% and 65%. By comparison, a 60 mV depolarization was seen in oat cell coleoptiles with microelectrode methods when the KCl concentration was increased from 1 to 25 mmol l1. This experiment was validated by assay with FC, a fungal toxin known to hyperpolarize plant cell membrane. Applied at 10 mmol l1, FC increased TPPþ uptake by 27% (2.8270.09 vs. 2.227 0.06 pmol mg FW1 in control; n ¼ 40 from two independent experiments). Taken together, these data suggest that membrane potential differences may be the main driving component for valine uptake, whereas it appears less important in the case of sucrose uptake.
Relative involvement of Hþ and Kþ in valine and sucrose uptakes Addition of sucrose to the tissues induced a very weak and transient modification of the pH in the incubation medium (DpH of 0.09 units, maximum in 30 min). By contrast, addition of valine led to a pH increase of 0.35 units, lasting up to 5 h (Fig. 7A). Such proton influx has been linked to a Hþ/ substrate co-transport mechanism and, by comparison, the results obtained here suggest that valine and sucrose uptakes occurred according to differ-
ent characteristics in the tap root of sugar beet. Titration of protons excreted in the bathing medium corroborated the weak and transient mobilization of Hþ during sucrose uptake compared to valine co-transport processes (Fig. 7B). Following this observation that sucrose absorption may not be exclusively dependent on the Hþ mobilization, we examined another line of research implying the involvement of a possible Kþ regulation. This line was considered in the light of the data obtained on KCl effects (Table 4) and in accordance with the Kþ-substrate co-transport mechanism presented by Van Bel and Van Erven (1979) in tomato internode tissues. The results in Fig. 8 show that modulators of Kþ mobilization, acting in different ways, affected sucrose uptake more strongly than valine uptake. Thus, LiCl known to antagonize Kþ ions inhibited valine and sucrose uptake by 18% and 35%, respectively. TEA, which acts on Kþ conductance, doubled inhibition of sucrose uptake compared with valine uptake. When Kþ availability was suppressed from the incubation medium, either by using choline chloride or Nmethyl-D-glucamine, significant sucrose inhibition of uptake was observed (respectively, 30% and 37%) whereas valine absorption was not significantly modified. It is therefore suggested that, unlike the case of valine uptake, Kþ may play a major role in sucrose uptake.
Discussion Numerous data concerning nutrient uptake have been obtained using leaf tissues, probably for
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Figure 8. Effect of modulators of Kþ mobilization (TEA, LiCl, choline chloride and N-methyl D-glucamine applied at 10 mmol l1) on the uptake of 1 mmol l1 valine (A) and 1 mmol l1 sucrose (B) by beet root slices. Pre-treatment lasted for 3 h and absorption for 30 min; data are means7SE (n ¼ 45) from three independent experiments.
experimental convenience. By contrast, the transport of nutrients in the tap root has been less studied, although this storage organ is clearly more appropriate than the leaf (source of photoassimilates) for the study of nutrient accumulation. In the tap root, sucrose is mainly accumulated at the base and in the middle part of the organ. Nutrients are mainly stored in pericyclic cells (Saftner et al., 1983). In these cells, sucrose is stored in the vacuole and can be released from this compartment only after hydrolysis (Leigh, 1984). In mature sugar beet root, sucrose unloading is apoplastic and uptake and accumulation in the vacuole occur without hydrolysis (Wyse, 1979). The data presented here demonstrate the ability of the storage roots of sugar beet to take up sucrose and amino acids from the apoplast. The contribution of this process to metabolite transport from sieve tubes to the parenchyma cells is yet to be determined, but by considering the root structure,
the possibility of unloading from sieve tubes into the apoplast still exists.
Amino acids in the economy of the taproot In the past, research has been mainly focused on the transport and accumulation of sucrose in these sink organs. In this respect, amino acids have been ignored. However, sugar beet roots contain many Ncompounds, mainly proteins, betaine and amino acids (Schiweck et al., 1993). In particular, amino acids have been found in the phloem sap obtained by exudation (Fife et al., 1962) and after collection by the aphid stylet technique (Lohaus et al., 1994). Until now, little has been known about the process governing the accumulation of amino acids in beet root storage cells. Therefore, this study focused on the uptake mechanism of valine; however, a comparative study on sucrose absorption was made
ARTICLE IN PRESS Valine and sucrose uptake in beetroot
in light of what had already been carried out by certain others, but changing somewhat their experimental conditions. Wyse (1979), Saftner and Wyse (1980), Saftner et al. (1983) have studied sucrose absorption at high external sucrose concentrations (40–100 mmol l1) and for long incubation times (3–8 h). Our experiments were generally conducted at a concentration of 1 mmol l1 and incubation time was generally restricted to 30 min. This last parameter was chosen because Saftner et al. (1983) have shown that sucrose uptake takes place primarily into a compartment having a t12 of 21 min, presumably the cytoplasm, whereas uptake in vacuole shows a longer turnover time. Concerning the experimental concentration, we chose the 1 mmol l1 value because Saftner et al. (1983) have shown that this value is in the range of the active component demonstrated for low concentrations (below 20 mmol l1) and also because it is near that of valine content estimated in the root exudate (Lohaus et al., 1994). This also raises the question of the actual substrate concentration in the apoplast in particular at the contact of the wall surrounding the storage cells after unloading from the phloem. It is not proven that the very high sucrose concentration measured in the sieve tube was found as such at the interface, so that a much lower concentration can be available for the cell uptake. In this direction, it has been estimated that sucrose concentration in the apoplast of broadbean leaf should vary between 1 and 4.5 mmol l1 (Delrot et al., 1983). These conditions at a lower concentration of sucrose can also be encountered in some deleterious environmental conditions in which the formation and translocation of sucrose can be hindered. This was observed following treatment with Cd, a common pollutant from various industrial productions (Greger and Lindberg, 1986), and in the presence of air pollutants O3 and SO2 (Okano et al., 1984). Variations in climatic conditions also affect nutrient partitioning as shown in chilling stress (Giaquinta and Geiger, 1973) and to a lesser extent with water stress (Sung and Krieg, 1979). In these conditions, the passive phase must become less preponderant and the increasing activity of a carrier must overcome this effect.
Similarities in the transport characteristics of valine and sucrose First, it has been shown that aging was also operating in these storage tissues and induced the same uptake modifications for the two substrates. These data indicate that, in this context, valine and
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sucrose uptake share identical properties. In particular, by considering the results of Marvier et al. (1997) on red beet storage tissue, it is suggested that enhancement of valine transport may be due to an increased plasma membrane HþATPase activity rather than to changes in carrier activity or to changes in metabolism. Second, our results specify the kinetics parameters calculated for valine uptake and our calculations enable them to be compared with those of Saftner et al. (1983). The uptake in both cases took place according to 3 phases as a function of the concentration. Two phases, from 0.1 to 1 mmol l1 and from 1 to 10 mmol l1, respectively, showed properties of high affinity, corresponding to the active component of transport, whereas for higher concentration values up to 100 mmol l1, the nonsaturating uptake is normally indicative of a diffusion process. This was corroborated by the observation that treatment with CCCP was not completely efficient at these high nutrient concentrations. Third, in contrast to this last assumption, valine and sucrose uptakes in the range of millimolar concentrations were strongly dependent on an active process as shown by CCCP action. In fact, CCCP is a metabolic uncoupler that increases the apparent proton conductance and reduces the electrochemical potential of Hþ across membranes. Valine and sucrose uptakes were very sensitive to treatment with CCCP inducing a reduction up to 75% upon application of the inhibitor. This high value underlines the sensitivity of nutrient transport in sink cells to variations of metabolism. Alkali cation-stimulated ATPase activity has been found in sugar beet root tissues (Lindberg, 1980; Briskin and Poole, 1983). Alkali cations stimulated active sucrose uptake considerably while ionophores, which act on electrochemical potentials of Hþ, Kþ and Naþ across membranes, strongly inhibited transport (Saftner and Wyse, 1980). The putative ATPases inhibitors DES and DCCD inhibited active valine and sucrose uptakes. Moreover, unlike Saftner et al. (1983), our results show that uptakes took place primarily at the plasma membrane since uptake inhibition was triggered following treatment with orthovanadate, whereas the process was not disturbed after treatment with nitrate known as a inhibitor of tonoplast ATPase activity (VATPases) (Chanson, 1993). This capacity of transport at the plasmalemma level was definitely demonstrated by the uptake of both substrates by PMV purified from beet root tissues. The use of PMV allows a direct study of the activity of the plasma membrane transport to be made without interference of the energizing primary process (Hþ
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pump ATPase) and without effect of metabolism. Under artificial pmf, both substrates are absorbed in PMV following an active mechanism since treatment with CCCP, that abolished this pmf, inhibited strongly this uptake. Fourth, sensitivity of tissues and PMV to PCMBS and mersalyl, nonpermeant sulfhydryl reagents, suggest that valine and sucrose influx involved membrane proteins with essential SH groups accessible to the medium.
Differences between the transport of valine and sucrose The comparative study carried out in our conditions has highlighted important differences in the way the two metabolites are absorbed. Firstly, it was observed that tap root tissues have the ability to absorb valine 4 times faster on a molar basis than they absorb sucrose. This result was expected since a similar observation was made in the leaf tissues (Sakr et al., 1993). A second point of importance comes from the observation on the long-term experiments: valine uptake became constant after 12 h absorption whereas sucrose was continuously absorbed, though at a reduced rate, after 20 h of uptake. This latter observation might be related to the reduced amount of sucrose available in the apoplast since, in similar experiments, Rosenkranz et al. (2001) have shown that induction of invertase activity takes place one day after wounding. A third interesting point is the shift noted in the optimum pH for uptake, which is more acidic for valine than for sucrose. However, this difference in optima between the two substrates is not surprising given the fact that they are transported by different transporters. Nevertheless, the sharp relation between sucrose uptake and pH in our experiment should be noted, differing fundamentally from the data of Wyse (1979) whose results were obtained at a 100 mmol l1 sucrose concentration. This result however resembles those of Saftner and Wyse (1980) who showed that the active sucrose uptake in the vacuole is optimum at pH 6.5 when the substrate concentration was lowered to 20 mmol l1 (i.e. in the active phase). Our work also suggests that the uptake of valine and sucrose are not sustained by the same driving force since membrane potential difference appears as the main component in valine uptake by contrast with sucrose uptake. This can be deduced from the observations that FC, which is known to induce membrane hyperpolarization, enhanced valine uptake (unlike sucrose absorption) and that high
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external KCl concentrations, resulting in membrane depolarization, hindered valine uptake but increased sucrose absorption. Such stimulation of sugar loading by a high external Kþ concentration has previously been described in Ricinus petioles (Malek and Baker, 1977) and in beet tap roots (Saftner and Wyse, 1980). Another major difference can be linked to the uptake process itself by considering the relative Hþ and Kþ mobilization following addition of the considered substrate. Thus, the large and longlasting Hþ influx following application of the amino acid to the tissues argues for an Hþ-substrate cotransport, whereas an Hþ involvement is more restricted in the case of sucrose uptake. By contrast, arguments can be put forward in favour of a major Kþ participation in the uptake of sucrose. Firstly, we observed that effectors of Kþ, either Liþ antagonizing Kþ ions movements or TEA, an inhibitor of Kþ channels, reduced sucrose uptake more greatly than valine uptake. This implies that, in the transport of sucrose, Kþ channels may be activated and have to be opened for charge exhange to maintain a constant electrical potential. Second, the starvation of external Kþ, achieved by the use of choline chloride and Nmethyl-D-glucamine, inhibited sucrose uptake to a greater extent. The fact that this effect was somewhat limited can be explained by the high amount of Kþ found in the apoplast, in particular in the pectinaceous cell wall (Saftner and Wyse, 1980). These data argue for the hypothesis that sucrose is co-transported in root-sink cells with Kþ (Saftner and Wyse, 1980) and can be included in a model in which Kþ ions are necessary for a ‘‘potassionated’’ uptake system presented by Van Bel and Van Erven (1979) to explain the loading of this nutrient in tomato internode discs.
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