JXB Advance Access originally published online on February 27, 2004
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Journal of Experimental Botany, Vol. 55, No. 398, pp. 879-888, April 1, 2004
© 2004 Oxford University Press
Regulation of Growth, Development and Whole Organism Physiology |
Sorbitol uptake is regulated by glucose through the hexokinase pathway in vegetative peach-tree buds
Received 13 October; Accepted 14 December 2003
1 UMR 547-PIAF Université Blaise Pascal, 24 avenue des Landais, F-63177 Aubière Cedex, France
2 UMR 547-PIAF INRA, domaine de Crouël, 234 avenue du Brézet, F-63039 Clermont-Ferrand Cedex 2, France
* To whom correspondence should be addressed. Fax: +33 4 73 40 79 16. E-mail: Soulaiman.SAKR{at}piaf.univ-bpclermont.fr
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Abstract |
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In peach trees (Prunus persica L. Batsch cv. Redhaven), sorbitol is a primary photosynthetic product and may play an important role in the budbreak process. Surprisingly, before budbreak (from January to early March), the concentration of sorbitol in the xylem sap decreases, while that of hexoses (glucose and fructose) increases. The aim of this work was to study the control of sorbitol uptake into vegetative buds by hexoses. Sorbitol uptake was selectively inhibited by hexoses at low and physiological concentrations and this effect was both reversible and concentration-dependent. In addition, the active uptake of sorbitol significantly declined in the plasma membrane vesicles-enriched fraction purified from glucose-treated vegetative buds, suggesting that the inhibitory action of glucose was at the membrane level. Finally, among several glucose analogues tested, only hexokinase substrates (2-deoxyglucose and mannose) were able to mimic the glucose effect, which was completely blocked by the hexokinase inhibitor mannoheptulose. These results represent the first steps towards a better understanding of polyol transport control in plants.
Key words: Budbreak, hexoses, regulation, sugar transport, vegetative bud, xylem sap.
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Introduction |
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The branching pattern of woody plants depends on the way in which the one-year-old shoots ramify and is, therefore, related to the budbreak capacity of the vegetative buds. In terms of sink–source relationships, a bursting vegetative bud may be regarded as a strong sink, as it does not produce carbohydrates (sugars) itself and hence can only burst and subsequently grow if it is suitably supplied with carbohydrates. When a vegetative peach-tree bud is out of endodormancy and able to burst under favourable growth conditions (from January to early March), its carbohydrate uptake capacity increases, concomitant with an increase in the expression and activity of plasma membrane H+-ATPase (Marquat et al., 1996; Aue et al., 1999; Gévaudant et al., 2001). These results support the hypothesis that budbreak requires a high import of sugars, essential to sustain bud growth. Since the budbreak occurs before the leafing period, the carbohydrates come mainly from the mobilization of products (i.e. starch) stored in the perennial parts of the tree (Bonhomme, 1998; Brunel, 2001).
The xylem pathway has been reported to play an important role in sugar export to the sink tissues in various perennial species such as apple (Malus domestica; Williams and Raese, 1974), walnut (Juglans regia; Ameglio et al., 2001; Lacointe et al., 2001), and sweet cherry (Prunus avium; McCamant, 1988, cited by Loescher et al., 1990). In the sweet cherry tree (a sorbitol-synthesizing plant like the peach tree), a detailed analysis of the xylem sap composition revealed that, besides sorbitol, hexoses (glucose, Glc and fructose, Fru), and sucrose (Suc) and its derivatives (raffinose and stachyose) are also transported in the xylem (McCamant, 1988, cited by Loescher et al., 1990). Furthermore, levels of Glc and Fru, but not of sorbitol or Suc, have been shown to be very high in sweet cherry sap before blooming. Although the significance of this hexose accumulation in the xylem sap is as yet unclear, it is well known that the role of hexoses in plants is multifunctional: they can act as an energy source, as precursors for metabolic processes, or as molecular signals regulating many vital functions (for a review see Sherson et al., 2003). In the present work, the focus was particularly on the signalling aspect.
In higher plants, soluble sugars (hexoses and Suc) are recognized as being important signalling molecules and are involved in many processes in the life-cycle of plants (Smeekens and Rook, 1997; Sheen et al., 1999; Gibson, 2000; Smeekens, 2000). Exogenous hexoses (Glc and Fru) significantly activate rubidium (Rb+) transport and water flux in the sunflower root system (Helianthus annuus; Quintero et al., 2001). A high concentration (close to 150 mM) of either Glc or Suc significantly reduces the expression of VfSut1, the Suc transporter in the broad bean seed (Vicia faba; Weber et al., 1997), while the Suc transporter in sugar beet (Beta vulgaris; Chiou and Bush, 1998) is down-regulated by its own substrate. The VvHT1 putative hexose transporter, which is expressed particularly during the ripening of grapes (Vitis vinifera), is also significantly up-regulated by relatively high concentrations (50 mM) of Glc (Atanassova et al., 2003). In contrast to this abundance of data relative to the regulation of soluble sugar transporters, nothing is known concerning the polyol transporter in plants. To date, the only data indicating a sugar control of polyol transport is obtained from a unicellular system. In a unicellular acidophilic red alga Galdieria sulphuraria (Oesterhelt and Gross, 2002), mannitol and sorbitol uptake are both selectively reduced by the presence of Glc or its analogues (MethylGlc, 3-O-MG) in the external medium.
In principle, to trigger sugar responses, soluble sugar signals have to be sensed in cells (Smeekens, 2000; Koch et al., 2000). One such sensing system is the hexokinase (HXK)-dependent pathway, similar to that already described in yeast and animal cells (Smeekens and Rook, 1997; Jang et al., 1997; Gibson, 2000; Smeekens, 2000). In plants, the HXK sensing system has been extensively studied and is known to control many important processes related to photosynthesis (Smeekens, 2000), germination (Pego et al., 1999), and polyol metabolism (Prata et al., 1997; Zamski et al., 2001). The central role of HXK in the regulation of such processes was demonstrated using metabolizable and non-metabolizable sugars. Thus, addition of a Glc analogue (3-O-MG) which cannot be phosphorylated was ineffective, whereas addition of 2-deoxyglucose (2-dGlc) or mannose (Man), two substrates for HXK, could mimic the Glc effect. Tests with exogenously applied mannoheptulose (Mhp) and analyses of transgenic Arabidopsis thaliana plants over- or under-expressing AtHXK provided further evidence for the role of HXK (Jang and Sheen, 1994, 1997; Jang et al., 1997; Xiao et al., 2000).
In the peach tree, sorbitol is, with Suc, a major photosynthetic product, which is transported along the phloem pathway to various sink tissues (Loescher et al., 1990; Noiraud et al., 2001a). There is growing evidence that sorbitol can also play a key role in many properties such as tolerance to abiotic stresses (Yancey et al., 1982; Le Rudulier et al., 1984; Loescher, 1987) and boron mobility within plants (Bellaloui et al., 1999). Sorbitol transport across the plasma membrane is catalysed by the H+/sorbitol symporter (Bouche-Pillon, 1994; Marquat et al., 1996, 1997). Although polyol transport has been receiving increasing attention over the past two years, for example, mannitol/H+ symporter and sorbitol/H+ symporter were identified in celery leaves (Apium graveolens; Noiraud et al., 2001b) and in sour cherry fruit (Prunus cerasus; Gao et al., 2003), respectively, its control remains poorly investigated in both the source and sink tissues of polyol-synthesizing plants.
As an initial step towards exploring the control of polyol transport in the sink tissues of higher plants, the effects of hexose on sorbitol uptake into vegetative peach-tree buds during the two months preceding budbreak (January and February) were studied. As a first result, it was found that the concentration of sorbitol in the xylem sap decreased (by a factor of three), whereas that of hexoses increased (by a factor of two) from January to the onset of budbreak (at the beginning of March). The possibility that sorbitol uptake into vegetative buds could be controlled by hexoses was therefore tested in the context of sugar sensing.
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Materials and methods |
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Plant material
One-year-old twigs of peach trees (Prunus persica L. Batsch cv. Redhaven) grown outside in an orchard at Clermont-Ferrand, France (45° N, 3° E, 350 m altitude) were sampled at regular intervals from January (breaking of dormancy) to early March 2002 (beginning of budbreak).
Xylem sap extraction and sugars analysis
Fourteen twigs (30 cm long) were collected monthly for xylem sap extraction. The xylem sap was collected by vacuum extraction according to Bollard (1953). To remove organic phosphate, amino and organic acids, xylem sap was purified using anion (DOWEX 50W 50*8 200–400 mesh form-H+) and cation exchange resins (BioRad AGI*8 100–200 mesh form-HCO-3). The purified fractions were dried under vacuum and resuspended in ultra-pure water. Soluble sugars were determined by HPLC as described by Gaudillère et al. (1992). Each sample was separated in an HPLC system consisting of a pre-column (BioRad Carbo C Micro Guard 30x4.6 mm) and a column (BioRad Aminex HPX 87C, 300x7.8 mm).
In vivo transport assays
In vivo uptake assays were carried out on vegetative buds collected in January and February. Because no difference was observed between the uptake measurements performed over these two months, data were pooled and the uptake data presented here are means of at least 15 buds from two independent experiments ±standard error (SE).
The uptake capacity of vegetative buds was studied as previously described by Marquat et al. (1999). Briefly, the freshly collected buds were incubated in control medium (20 mM MES/NaOH pH 5.0, 0.25 mM CaCl2, 0.5 mM MgCl2) to which 1 mM labelled sorbitol ([U-14C] sorbitol 11 kBq ml–1) or 1 mM labelled Val ([U-14C] Val 11 kBq ml–1) were added. After 1 h of incubation, apoplastic label was removed by three washes of 3 min in control medium. Each bud was separately weighed and then lysed for 16 h in a lysis medium (60 µl of HClO4/H2O2, 1:2, v/v). The radioactivity was measured in a liquid scintillation analyser (1600 TR, Packard).
Depending on the experiment, either 10 mM of each sugar detected in the xylem sap (see Table 1 in the Results), hexoses at concentrations ranging from 0.5–10 mM (see Fig. 2 in the Results) or 10 mM of different analogues of Glc (see Table 2 in the Results) was added to the control medium.
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In order to check whether the Glc-induced inhibitory effect on sorbitol uptake was reversible, 20 buds were collected and divided into four groups of five buds. The first group was incubated on control medium for 1 h with 1 mM [U-14C] sorbitol alone ([14C] sorbitol). The second group was incubated on control medium with both 1 mM [U-14C] sorbitol and 10 mM Glc ([14C] sorbitol+Glc). The third group was first placed for 1 h on control medium that contained 1 mM non-labelled sorbitol, washed (3x3 min) and then incubated for an additional hour in control medium with both 1 mM [U-14C] sorbitol and 10 mM Glc (sorbitol/[14C] sorbitol+Glc). The last group was first placed for 1 h on control medium containing 10 mM Glc, then washed (3x3 min) and incubated for an additional hour with 1 mM [U-14C] sorbitol (Glc/[14C] sorbitol).
Sorbitol metabolism in vegetative buds
Metabolism of [U-14C] sorbitol was measured in vegetative buds that had been incubated for 1 h with 1 mM [U-14C] sorbitol, in the presence or absence of 10 mM Glc. Sugars were extracted as described by Gaudillère et al. (1992) and separated by HPLC as described above.
Plasma membrane vesicles (PMV)-enriched fraction isolation and immunoblotting
Three-hundred vegetative buds were used for the PMV-enriched fraction extraction. The PMV-enriched fraction was purified by phase partitioning as initially described for vegetative peach-tree buds by Aue et al. (1999) and then stored as a concentrated protein suspension (6–10 mg protein ml–1) in KP buffer (50 mM potassium phosphate buffer pH 7.5, 330 mM Suc, 0.25 mM CaCl2, 0.5 mM MgCl2) in the presence of 0.5 mM dithiothreitol (DTT).
Three kinds of PMV-enriched fractions were isolated: the first was directly isolated from freshly collected vegetative buds and the other two were isolated from buds placed for 1 h in control medium, either with 10 mM Glc (PMV-enriched fraction from Glc-treated buds) or without Glc (PMV-enriched fraction from Glc-untreated buds).
Plasma membrane H+-ATPase is commonly used as a marker of plasma membrane (for a review see Palmgren, 2001). To assess the enrichment of PMV in the enriched fraction from Glc-treated buds and Glc-untreated buds, the level of plama membrane H+-ATPase was compared by immunoblot test (Aue et al., 1999). A purified polyclonal antiserum raised against a conserved peptide of H+-ATPase (CDPKERAGIREVHF) diluted 1/3000 was used as primary antibody. An anti-rabbit IgG (H+L) (PARIS, 1/10000 dilution) was used as secondary antibody. The protein–antibody complex was detected with a chemiluminescence protein gel blotting detection system (ECL, Amersham Pharmacia Biotech) and exposed for 5–15 min to X-ray films.
In vitro transport assays
The PMV-enriched fraction was used to measure active uptake (total uptake minus passive uptake) of sorbitol and Val as previously described in Salmon et al. (1995). Total uptake in energized conditions (
pH+
) was initiated by mixing 10–15 µg PMV-enriched fraction (about 2 µl) with 400 µl of NaP buffer (50 mM sodium phosphate buffer pH 5.5, 330 mM Suc, 0.25 mM CaCl2, 0.5 mM MgCl2) to which 5 µM valinomycin and [U-14C] sorbitol (11 kBq ml–1) or [U-14C] Val (11 kBq ml–1) were added. The combination of the KP buffer (50 mM potassium phosphate buffer pH 7.5, 330 mM Suc, 0.25 mM CaCl2, 0.5 mM MgCl2) inside the vesicles with the NaP buffer in the incubation medium creates a pH and a (Lemoine and Delrot, 1989). For passive uptake in non-energized conditions (pH==0), the incubation medium was KP buffer to which [U-14C] sorbitol (11 kBq ml–1) or [U-14C] Val (11 kBq ml–1) was added. The incubation was stopped at selected times by the addition of 2x2 ml of washing medium (chilled NaP buffer with 1 mM HgCl2 for total uptake, or KP buffer with 1 mM HgCl2 for passive uptake) and the radioactivity was measured in a liquid scintillation analyser (1600 TR, Packard). The concentration of protein in each PMV-enriched fraction was spectrophotometrically determined by the Bradford method (Bradford, 1976) in the presence of 0.01% (v/v) Triton X100. Bovine serum albumin was used as a standard.
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Results |
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Dynamics of total carbon, sorbitol, and soluble sugar concentrations in the xylem sap
The amount of sap extracted from the xylem was almost constant from January to the beginning of March. Sap total carbon concentration slightly decreased in February (by 20% compared with the January value) and increased again in March. Among the various sugars detected in peach xylem sap, only Glc, Fru, and sorbitol exhibited monthly variations (Fig. 1). Levels of Suc, raffinose, and stachyose were very low (respectively 9%, 2%, and 1% of total sugars) over this period.
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In January, sorbitol was the predominant sugar in the xylem sap (close to 60% of total sugars) and its concentration was about 4-fold higher than those of Glc and Fru. In February and March, levels of sorbitol decreased significantly (by a factor of three as compared to January). Conversely, concentrations of Glc and Fru were lowest in January, began to increase slightly in February and peaked in March (about 2–3-fold higher than in January).
Effects of the soluble sugars in xylem sap on sorbitol uptake into vegetative buds
Preliminary experiments showed that the uptake of sorbitol by vegetative buds was linear for at least 2 h (K Maurel, unpublished data) and hence an incubation time of 60 min was chosen. Various sugars detected in the xylem sap were tested at a physiological concentration of 10 mM (Jang and Sheen, 1994; Quintero et al., 2001), for their effects on sorbitol uptake by vegetative buds collected in January and February (during the two months preceding budbreak).
As shown in Table 1, sorbitol uptake was only weakly inhibited by raffinose and stachyose (approximately 20%), but was strongly inhibited by it own substrate, Glc and Fru (approximately 80%). Suc was far less effective than hexoses as it reduced sorbitol uptake by only about 30%.
On the other hand, a chromatographic analysis showed that in vegetative buds incubated with or without 10 mM Glc, less than 2% of all the [U-14C] sorbitol absorbed had been converted into Glc after 1 h.
Effects of Glc and Fru on Val and sorbitol uptake into vegetative buds
In order to determine whether the inhibitory effect of hexoses (Glc and Fru) was selective for sorbitol uptake, sorbitol and Val uptakes were compared for sensitivity to hexoses. Val is also taken up in plants by an H+/substrate symporter (Bush, 1993; Delrot et al., 2000).
The Val uptake was insensitive to hexoses even when the buds were incubated with 10 mM Glc or Fru (Fig. 2A, B). By contrast, low concentrations of Glc were sufficient to reduce the sorbitol uptake, which decreased to 54% in the presence of 1 mM Glc. This inhibition increased with the Glc concentration to reach a maximum at 5 mM Glc (86% versus control) and no further inhibition was observed at 10 mM Glc (Fig. 2A). Addition of Fru to the external medium resulted in an effect similar to that of Glc, with a maximum inhibition at 10 mM (Fig. 2B).
Since Glc and Fru had comparable inhibitory effects on sorbitol uptake, for simplicity only Glc was employed in the following experiments.
Effects of Glc on active uptake of sorbitol into the PMV-enriched fraction purified from freshly collected vegetative buds
As sorbitol is the reduced form of Glc, one possible explanation for these results was that Glc might inhibit sorbitol uptake by competing for the sorbitol transporter. In order to check this possibility, the impact of sorbitol and Glc on the active uptake of sorbitol (total uptake minus passive uptake) into the PMV-enriched fraction purified from freshly collected vegetative buds was studied. The sorbitol effect on its own uptake into the PMV-enriched fraction is used as a positive control.
As shown in Fig. 3A, the active uptake of sorbitol rapidly reached its maximum after incubation of 1 min and decreased over the next 4 min. This indicates that sorbitol must be actively absorbed into the PMV-enriched fraction and fits previous reports relating to vegetative buds (Marquat et al., 1999). In subsequent experiments concerning the active uptake of solutes into the PMV-enriched fraction, an incubation time of 1 min was chosen to give an estimate of the initial velocity of uptake (Fig. 3A; Sakr et al., 1993; Salmon et al., 1995, and references therein). Moreover, this active uptake of sorbitol was not inhibited by the presence of 10 mM Glc for 1 min in the uptake medium (Fig. 3B). These data are different from that obtained with 10 mM sorbitol, which strongly inhibited its own uptake into the PMV-enriched fraction (Fig. 3B). Hence the inhibitory effect of Glc on sorbitol uptake into vegetative buds was not merely due to competition between the two sugars for the sorbitol transporter.
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Effects of Glc on active uptake of sorbitol into the PMV-enriched fraction purified from Glc-treated vegetative buds
To check whether the effect of Glc on sorbitol uptake occurs at the membrane level, parallel experiments were run using the purified PMV-enriched fraction prepared from vegetative buds placed for 1 h in a control medium containing 10 mM Glc (PMV-enriched fraction from Glc-treated buds) or no Glc (PMV-enriched fraction from Glc-untreated buds). The enrichment of these two PMV-enriched fraction preparations was verified with purified antibodies raised against the central domain of plasma membrane H+-ATPase. These antibodies cross-reacted only with a single band at 97 kDa, in PMV-enriched fraction or the corresponding microsomal fractions (MF) (Fig. 4A), which is consistent with the classical size of H+-ATPase (Arango et al., 2003). Furthermore, comparison of the intensities of the H+-ATPase band in the PMV-enriched fraction and their corresponding MF showed that plasma membrane H+-ATPase was strongly enriched in the PMV-enriched fraction (Fig. 4A). These data are in accordance with those initially reported for PMV from vegetative peach-tree buds (Aue et al., 1999), indicating that the PMV-enriched fractions were pure. Moreover, the intensity of the H+-ATPase band was not significantly different between the PMV-enriched fraction from Glc-treated buds and the PMV-enriched fraction from Glc-untreated buds (Fig. 4A).
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The active uptakes of sorbitol and Val into these two kinds of PMV-enriched fraction were measured after incubation for 1 min. As seen in Fig. 4B, the active uptake of sorbitol into the PMV-enriched fraction from Glc-treated vegetative buds was 2-fold lower than that into PMV from Glc-untreated vegetative buds, whereas the uptake of Val was almost equal, independent of Glc treatment. Therefore, the inhibitory effect of Glc on active sorbitol transport did not result from a general modification of the plasma membrane proteins, but was specific for sorbitol transport.
Reversibility of the inhibitory effect of Glc on sorbitol uptake into vegetative buds
It has recently been shown that Suc-dependent changes in the Suc symporter from Beta vulgaris were reversible (Chiou and Bush, 1998). Therefore, it was tested whether the Glc effect on sorbitol uptake was also reversible. When buds treated with Glc-free medium were transferred for an additional 1 h to control medium containing both 1 mM [14C] sorbitol and 10 mM Glc (sorbitol/[14C] sorbitol+Glc), the sorbitol uptake was 2-fold lower than in vegetative buds directly incubated with 1 mM [U-14C] sorbitol alone ([14C] sorbitol) (Fig. 5). Furthermore, this inhibition (about 50%) was similar to that observed when vegetative buds were directly incubated with both 1 mM [U-14C] sorbitol and 10 mM Glc ([14C] sorbitol+Glc). Conversely, the inhibitory effect of Glc on sorbitol uptake was almost abolished when vegetative buds previously incubated with 10 mM Glc alone were transferred for an additional 1 h to a Glc-free medium containing 1 mM [14C] sorbitol alone (Glc/[14C] sorbitol) (Fig. 5).
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Effects of Glc analogues and Mhp on sorbitol uptake into vegetative buds
Soluble sugar levels have been reported to mediate changes in a variety of metabolic and developmental processes in plants and HXK could play an essential role in these pathways. In this context, the impact of Glc analogues on sorbitol uptake by vegetative buds was studied first (Table 2). Substrates of HXK which are transported into the cell and phosphorylated were able to mimic the inhibitory effect of Glc. Man or 2-dGlc 10 mM decreased sorbitol uptake significantly (71% and 41%, respectively). On the contrary, under the same conditions, a Glc analogue which is not a substrate of HXK (3-O-MG) was ineffective. The efficacy of Man and 2-dGlc in inhibiting sorbitol uptake highlights the potential importance of sugar phosphorylation in signal transduction mechanisms mediated by HXK. If the latter is indeed involved in the blockage of sorbitol uptake, the effect should be reversed by Mhp which is a specific inhibitor of HXK. Thus, the sorbitol uptake of vegetative buds incubated with 10 mM Glc was determined in the presence or absence of Mhp at concentrations of 10 mM and 20 mM. As seen in Table 2, Mhp reversed the inhibitory effect of Glc in a concentration-dependent manner, while Mhp alone (10 or 20 mM) did not affect sorbitol uptake.
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Sorbitol uptake is selectively, but not competitively, inhibited by Glc
The regulation of polyol transport by sugars in plants has only recently been described and indeed the only published data indicating an inhibition of sorbitol uptake by exogenous Glc are recent and relate to a unicellular alga (Oesterhelt and Gross, 2002). This work shows that Glc also has an inhibitory effect on sorbitol uptake in vegetative peach-tree buds (Fig. 2A; Tables 1, 2). The Glc concentrations (0.5–10 mM) required to inhibit sorbitol uptake were similar to those used in other studies (Jang and Sheen, 1994; Quintero et al., 2001). It is noteworthy that Val uptake, which is also catalysed by symporter H+/amino-acids, was insensitive to Glc under the same experimental conditions (Fig. 2A). This means that the action of Glc on sorbitol uptake is selective and does not result from an indirect effect on cell metabolism. Such a conclusion is further supported by the fact that no significant difference in sorbitol uptake was found between vegetative buds directly incubated with 1 mM sorbitol ([14C] sorbitol) and buds pre-incubated for 1 h with 10 mM Glc and then transferred to a Glc-free medium containing 1 mM [14C] sorbitol (Glc/[14C] sorbitol) (Fig. 5). The selective Glc effect has also been demonstrated in previous transport studies in plants (Quintero et al., 2001) and yeast (Ramos et al., 1992; Alijo and Ramos, 1993). Moreover, since an iso-osmotic solution of 3-O-MG did not inhibit sorbitol uptake (Table 2), the inhibition did not merely result from an osmotic effect. Finally, as sorbitol inhibited its own uptake into the PMV-enriched fraction (70%), but Glc did not inhibit sorbitol uptake into the same PMV-enriched fraction (Fig. 3B), it can be concluded that this inhibition was not of the competitive type.
Sorbitol uptake is also inhibited by Fru
Data from two independent experiments (Table 1; Fig. 2B) show that Fru also inhibited sorbitol uptake in a selective and concentration-dependent manner, to a similar extent as Glc (about 80%). This finding of an inhibitory effect of Fru, comparable to that of Glc, is consistent with previous results obtained in plants (Graham et al., 1994; Quintero et al., 2001) and yeast (Pernambuco et al., 1996). Hence only the effect of Glc was investigated in more detail.
All the results presented above (Figs 2A, B, 3B; Table 1) support the idea that hexoses are strong inhibitory regulators of sorbitol uptake in vegetative buds. Therefore, it is suggested that the apoplastic sorbitol supply to the bursting vegetative buds might be reduced by hexoses. If this is true, the bursting buds could preferentially absorb hexoses rather than sorbitol and it will be of interest to examine the respective roles of sorbitol and hexoses in the budbreak process.
Sorbitol uptake is inhibited by Glc at the membrane level
Sorbitol uptake into vegetative buds may depend not only on its transport through the plasma membrane, mediated by an H+/sorbitol symporter, but also on its metabolism. Therefore, the inhibition of sorbitol uptake by Glc could be explained on the basis of a ‘detrimental’ effect on sorbitol-metabolizing enzymes. Sorbitol is metabolized by an NAD-dependent sorbitol dehydrogenase (NAD-SorDH) and an oxidase (SorOX), which convert it into Fru or Glc, respectively (Loescher et al., 1982; Lo Bianco and Rieger, 2002). Nothing is yet known about the regulation of these enzymes by sugars and the only published data concern the mannitol-catabolizing enzyme mannitol dehydrogenase (MTD), which catalyses the oxidation of mannitol to Man. This enzyme was inhibited after incubating cultured celery cells with 20 mM Glc for the relatively long time of 12 h (Prata et al., 1997). In the present experiments, the possibility that the inhibition of sorbitol uptake by Glc might reflect changes in sorbitol metabolism can, nevertheless, be ruled out because only 2% of all the radiolabelled sorbitol taken up in the presence or absence of 10 mM Glc was converted into Glc. Hence it is probable that Glc has little effect on the sorbitol metabolism of vegetative buds, at least within the short incubation time of 1 h.
Alternatively, Glc could directly affect sorbitol transport, irrespective of any impact on its metabolism. One approach to test this hypothesis was to look at the effects of Glc at the membrane level using purified PMV-enriched fraction. The PMV-enriched fraction generally provides a reliable image of the changes in solute transport observed at a physiological level (Sakr et al., 1993, 1997; Roblin et al., 1998; Chiou and Bush, 1998). As an example, to determine the effects of Suc on Suc transporters in sugar beet, PMV-enriched fractions were purified from leaves incubated or not with Suc and then used for uptake assays (Chiou and Bush, 1998). The uptakes of Suc and other solutes (alanine and Glc) were compared and since only the Suc transporter activity declined in the PMV-enriched fraction from Suc-infiltrated leaves, these authors concluded that the Suc transporter is regulated by its own substrate. In the same way, experiments were performed using the PMV-enriched fraction prepared from vegetative buds treated with Glc or Glc-free medium. Glc clearly inhibited sorbitol uptake at the membrane level (Fig. 4B). Indeed, the active uptake of sorbitol measured in the PMV-enriched fraction from Glc-treated buds dropped to 50% of that measured in control (PMV-enriched fraction from Glc-untreated buds). By contrast, the active uptake of Val did not respond to Glc treatment (Fig. 4B). These results for sorbitol and Val uptake into the PMV-enriched fraction were, moreover, in full accordance with those obtained using vegetative buds (Table 1; Fig. 2A).
In summary, the fact that the active uptake of sorbitol was significantly reduced in the PMV-enriched fraction from Glc-treated vegetative buds indicates that generic changes in membrane integrity cannot account for the inhibition of sorbitol transport activity and that the effect of Glc is most likely specific for the sorbitol transport(er). This conclusion is further supported by the fact that levels of plasma membrane H+-ATPase were the same in the two kinds of the PMV-enriched fraction (Fig. 4A). One possible explanation is that Glc could induce post-transcriptional and/or post-translational regulation of the sorbitol transporter, as previously reported for other sugar transporters (see Introduction). Further experiments, aiming in this case at the characterization of a cDNA encoding the sorbitol transporter and its upstream regulatory sequence in the vegetative peach-tree bud, will be required to elucidate how hexoses regulate sorbitol transporter(s).
HXK mediates the inhibition of sorbitol uptake
The experiments described here (Table 2) indicate that the specific inhibitory effect of Glc on sorbitol uptake is probably mediated by the HXK-dependent pathway. This conclusion is based on four lines of evidence: (1) Man, which is phosphorylated by HXK, was able to inhibit sorbitol uptake to the same extent as Glc (about 70%); (2) 2-dG, which is a substrate of HXK but is very weakly metabolized by glycolysis (Klein and Stitt, 1998), also led to the inhibition of sorbitol uptake, but to a lesser extent than Glc (41% versus 79%); (3) 3-O-MG, which is not phosphorylated by HXK (Sheen et al., 1999), or is phosphorylated to 3-O-MG-6 phosphate but not perceived as a sugar in maize roots (Zea mays; Cortès et al., 2003), only had a marginal effect on sorbitol uptake (18%, Table 2); (4) Mhp (20 mM), which is a specific inhibitor of HXK, completely blocked the effect of Glc. In addition, the Glc-induced changes in sorbitol transport were reversible (Fig. 5), suggesting that the HXK pathway may modulate the transport activity as a function of the flux through the HXK reaction.
On the other hand, a number of observations support the view that the effects of Suc on sorbitol uptake cannot be mediated by the Suc-specific pathway. Firstly, at the same molar concentration (10 mM), Suc induced a much weaker inhibition of sorbitol uptake than Glc (30% versus 80%). Secondly, the effects of Suc could be mimicked by a Glc concentration of 0.5 mM (Fig. 2A). Thirdly, Suc is likely to be converted into hexoses (Glc and Fru) by the extracellular invertase of vegetative buds and, therefore, the inhibitory effect of Suc is due to hexoses (Glc and Fru) rather than to Suc itself, as was also found in other studies (Weber et al., 1997; Jang et al., 1997; Ehness et al., 1997). Overall, the data here and in previous reports (Prata et al., 1997; Zamski et al., 2001) indicate that HXK would be of major importance in regulating the metabolism and transport of polyols in polyol-synthesizing plants. Based on the data of Fig. 5 (i.e. Glc effect is reversible) and Table 2 (i.e. involvement of HXK), the flux through the HXK reaction may be important in initiating the signal which ultimately leads to the selective inhibition of sorbitol transport. A similar scenario has previously been proposed by Graham et al. (1994).
In conclusion, the present study provides evidence that sorbitol uptake is inhibited by exogenous hexoses (Glc and Fru) in vegetative peach-tree buds. This inhibition was found to be concentration-dependent, reversible, specific for sorbitol transport, and mediated by the HXK signalling pathway. Further experiments will now be required to elucidate signal transduction cascades that result in this specific inhibition of sorbitol uptake by hexoses.
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Acknowledgements |
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Authors are grateful to Dr Rémi Lemoine (Unité Mixte de Recherche-Centre National de la Recherche Scientifique 6161, Transport des Assimilats, Laboratoire de Physiologie, Biochimie et Biologie Moléculaires Végétales, Poitiers, France) for critically reading the manuscript. This work was supported by the Ministère de la jeunesse, de l’éducation nationale et de la recherche and INRA.
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