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Dormancy in vegetative buds of peach: relation between carbohydrate absorption potentials and carbohydrate concentration in the bud during dormancy and its release
Scientia Horticulturae 79 (1999) 151±162
Dormancy in vegetative buds of peach: relation between carbohydrate absorption potentials and carbohydrate concentration in the bud during dormancy and its release Christel Marquata, Marc Vandammeb, Michel Gendrauda, Gilles PeÂtela,* a Unite AssocieÂe Bioclimatologie - P.I.A.F. (INRA - Universite Blaise Pascal), Physiologie et GeÂneÂtique veÂgeÂtales, 24 avenue des Landais, 63177, AubieÂre CedeÂx, France b INRA-Station de Bioclimatologie, Domaine de Crouelle, 63069, Clermont-Ferrand Cedex, France
Accepted 31 July 1998
Abstract The absorption of sucrose and sorbitol by the bud and the stem of Prunus persica was evaluated during the rest period. The utilization of p-chloromercuribenzenesulfonic acid (PCMBS), an inhibitor of sucrose transporter, allowed estimation of the active absorption of sucrose in different isolated organs (bud, bud's underlying tissues and stem). It was assumed that the sink capacities of tissues depended on their potential to absorb carbohydrates by active transport. Endogenous carbohydrate levels in the tissues were also measured. In the bud, the qualitative and quantitative distribution of carbohydrates could be related to the absorption potential of the bud. During dormancy, the bud exhibited a low absorption potential and increased its sucrose concentration by starch hydrolysis. Soluble sugars accumulated during winter. During dormancy release, the bud was able to absorb carbohydrates allowing carbon storage. The data presented led to the proposal of a scheme of events occurring during bud dormancy and its release. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Vegetative bud; Absorption potentials; Sucrose; Sorbitol; Carbon storage
* Corresponding author. Fax: +33-473-407934. 0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 8 ) 0 0 2 0 3 - 9
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1. Introduction Dormancy can have three causes according to Lang et al. (1987): (i) an endogeneous signal within the affected structure (endodormancy); (ii) a biochemical signal originating in a structure other than the affected structure (paradormancy); (iii) environmental factors which affect over all plant metabolism (ecodormancy). The factors which control the induction and duration of dormancy have not been elucidated until now. In Prunus persica (L.) Batsch, the induction of vegetative bud dormancy takes place during late August and budburst occurs at the end of February. The state of rest could be linked, at least partially, to trophic correlations between the bud's underlying tissues and the bud itself, thus regulating its growth (paradormancy). In peach, during the bud's rest period, absorption of nutrients by the bud's underlying tissue and the stem could lead to the bud's deprivation and reduce its ability for growth (Gendraud and PeÂtel, 1990). Our work aims to identify (i) the absorption potential of the bud and the stem, in Prunus persica, during the rest period and during dormancy release; (ii) the variation in the major soluble sugars (sucrose, fructose, glucose, sorbitol and raffinose stachyose) and starch in the stem and the bud. Active absorption of sucrose was measured by the use of PCMBS (p-chloromercuribenzenesulfonic acid) to inhibit the sucrose transporter (M'Batchi and Delrot, 1984; Sakr et al., 1993), while allowing simple diffusion. The effect of this inhibitor was previously evaluated in situ on the organs of single node cuttings (Marquat et al., 1997). These two methods allowed the bud's dormancy to be related to its sugar absorption potential by active transport and to the level and the use of its carbohydrate content. 2. Material and methods 2.1. Plant material Current year twigs were sampled from 10 year-old peach trees (Prunus persica L.). Budburst occurred at the end of February or at the beginning of March. Only the axillary and isolated vegetative buds were used. For the estimation of absorption potentials, 3 samples were used for each organ (scaled bud and stem) from October to January and 5 different measurements were made on each sample. For the measurement of soluble sugars and starch levels, 50 scaled buds and stem pieces were collected every month from October to January. They were stored at ÿ808C, then lyophilized. Dried samples were ground to a fine powder and weighed.
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2.2. Determination of solute uptake by different tissues of peach Each isolated organ (scaled bud, stem discs) was weighed and immersed, for 2 h, in 400 ml of a medium containing 300 mM mannitol, 20 mM Mes-KOH (pH 6.5), 0.5 mM CaCl2, 0.25 mM MgCl2, either in the presence or in the absence (control) of 1 mM PCMBS. Mannitol concentration was changed to maintain a constant osmolarity. PCMBS was used in order to evaluate `active' absorption. Samples were rinsed with a control medium for 30 min and then immersed in a control medium labelled with 300 mM [3 H] mannitol (specific activity 0.97 TBq mmolÿ1; Sigma, USA). Uptake was initiated by adding 1 mM [14 C] sucrose (specific activity 23 GBq mmolÿ1; Amersham, UK) or 1 mM [14 C] sorbitol (specific activity 12 GBq mmolÿ1; Amersham, UK) and run at 238C for 3 h. Shorter times (1 h, 2 h) were tested but were insufficient, as 3 h corresponded to the time necessary to obtain the diffusion equilibrium. Samples incubated with [3 H] mannitol allowed us to determine the free space. As mannitol does not diffuse into the intracellular medium and is not transported, it allowed the incorporation of sucrose into the intracellular medium to be evaluated. At diffusion equilibrium, the 14 C=3 H ratio was compared with the initial 14 C=3 H ratio in the medium (0.5). After this period, each tissue was rinsed with the same control medium, dried, and homogenized using a mortar. Labelled sugars were extracted from the homogenates in 80% ethanol for 2 min at 1008C. Radioactivity was measured by scintillation counting in the presence of 5 ml liquid scintillation cocktail. This experiment allowed evaluation of the sucrose transporter's specificity for sucrose and the protecting effect of the sucrose on PCMBS inhibition. 2.3. Sugar extraction We followed the procedure described by GaudilleÁre et al. (1992). Seven mg of dried material (scaled buds 2 mm in length and stem discs 4 mm in diameter and 1 mm thick) were used for analysis. Soluble sugars were extracted with boiling 80% ethanol for 30 min. Extracts were centrifuged at 15 000g for 10 min in order to remove insolubles (starch). This operation was repeated twice. The alcohol fraction containing the soluble sugars was evaporated to dryness under vacuum. The pellet was solubilized in 500 ml of ultra-pure water. Soluble sugars were purified using anion- (DOWEX 50 W 50*8 200±400 mesh form-H) and cation-exchange (BIORAD AGI*8 100±200 mesh form HCO3) resins to remove organic phosphate, amino acids and organic acids. The purified extracts were dried for 12 h under vacuum and were then resuspended in 500 ml of H2O.
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2.4. Carbohydrate analysis Soluble carbohydrates were determined by HPLC. One hundred ml of extract was filtered on Millipore filter (0.45 mm) adapted on a syringe. Twenty ml of each extract was separated in an HPLC system consisting of a pre-column (BIORAD Carbo P Micro Guard 30 4.6 mm) and a column (BIORAd Aminex HPX 87 P 300 7.8 mm). The carbohydrates were quantified from peak area calculations related to regression curves of standards. Sorbitol content was measured by an enzymatic method (Boehringer-Mannheim) based on the following enzymatic reactions (Kunst et al., 1984 and Loescher, 1987): sorbitol NAD
!
sorbitol dehydrogenase
fructose NADH H
(1)
To avoid reversion of this first reaction, the (NADH H) obtained is transformed to NAD according to Eq. (2). This also ensured the complete hydrolysis of sorbitol: NADH H pyruvate
!
lactate dehydrogenase
lactate NAD
(2)
The fructose obtained from Eq. (1) is then transformed to glucose-6-phosphate, as follows: fructose ATP ! fructose-6-phosphate
(3)
fructose-6-phosphate ! glucose-6-phosphate
(4)
The last reaction, catalysed by the glucose-6-phosphate dehydrogenase, leads to the formation of NADPH H: glucose-6-phosphate NADP ! gluconate-6-phosphate NADPH H (5) (NADPH H) formation was quantified at 340 nm and corresponded to the quantity of oxidised sorbitol (Eq. (1)). The pellets were dried under vacuum for 30 min, solubilized in 1 ml NaOH 0.02 N and placed at 908C for 30 min in order to remove cell walls and to solubilize the starch. 50 ml of the suspension were incubated with 100 ml of citrate buffer (pH 4.2) containing 15 units of amyloglucosidase (Sigma) for 30 min at 508C and centrifuged at 15 000g. The glucose released was quantified by measuring the formation of NADPH H at 340 nm (Kunst et al., 1984), according to the following reaction: glucose ATP ! glucose-6-phosphate The glucose-6-phosphate was then transformed to gluconate-6-phosphate, according to Eq. (5), inducing the formation of NADPH H.
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3. Results 3.1. Sucrose absorption Total sucrose absorption increased between September and October in both the stem and the bud. In the bud, total absorption decreased in December (Fig. 1(a)). In the stem, total sucrose absorption decreased a little from November (Fig. 1(b)). Active absorption decreased from October in both tissues. In December, both
Fig. 1. Changes in active (&) and total (&) absorption of sucrose in the bud (a) and in the stem (b). Each experiment was conducted on 3 different samples. Data are means S.E. of 5 measurements from 3 different samplings.
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total and active uptake were higher in the stem than in the bud. The stem values did not differ from the values found in October. In January, the ability of the bud to accumulate sucrose increased markedly and active absorption reached a maximum. In the stem, active absorption was also important in January. Globally, an enhancement of active sucrose absorption was observed in January. 3.2. Sorbitol absorption Sorbitol absorption was not inhibited by PCMBS. So, study of active absorption of sorbitol in the different organs was not possible (Marquat et al., 1997). Variation in sorbitol absorption potential was less for sucrose (Fig. 2). In the stem, sorbitol absorption reached a minimum in November and a maximum in December. In the bud, absorption was low in November and December, then increased in January. In December, the stem exhibited an important sink strength for sorbitol. In January, absorption potentials were comparable in the stem and in the bud. As no experiments were previously monitored using vegetative buds of peach, soaking scaled buds in sugar solution may not constitute a valid simulation of the actual transport of sugars from the stem into buds. So, an alternative method was tested in which the cut end of a single node cutting was placed on the sugar solution. Under these conditions, total absorption of sucrose and sorbitol in the buds was measured. Similar results were observed using both methods. For instance, in October, total absorption of sucrose was 1.4 nmol/mg FW using the single node cutting method and 1.3 nmol/mg FW using the soaked scaled buds
Fig. 2. Changes in total absorption of sorbitol in the bud (&) and in the stem (&). Each experiment was conducted on 3 different samples. Data are means S.E. of 5 measurements from 3 different samplings.
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method. The same observation was made concerning sorbitol absorption. Unfortunately, the single node cutting method could not be used to measure the active absorption of sugars, requiring the use of PCMBS. Experiments showed that PCMBS action, in this condition, was not reproducible enough to obtain useful data. Consequently, the soaked scaled buds method, reflecting the actual absorption of sugars from stem to buds through vascular bundles was chosen. 3.3. Carbohydrate contents of buds and stem Whatever the month, the buds had a higher level of total soluble sugars (three times that of the stem) (Fig. 3). In the stem, 50% of starch was hydrolysed between October and December (Fig. 3). From December, starch reserves increased (5 mg/g DW) and soluble sugars decreased (9 mg/g DW). In the bud, starch was hydrolysed from October until December. From December, starch level increased in the bud and soluble sugars level decreased by a similar amount (30 mg/g DW). In November, stachyose and raffinose were detected in both the stem and the bud (Fig. 4(a)). In the stem, their concentration progressively decreased until February. In the bud, stachyose raffinose levels increased to a high value in January. In the stem, sucrose and sorbitol levels were constant (Fig. 4(b) and (c)). Fructose and glucose levels increased until December, then decreased until budburst. In the bud, sucrose and sorbitol represented 78.5% of the total soluble sugars. Between October and December, the sucrose level increased and glucose (Fig. 4(d)), fructose (Fig. 4(e)) and sorbitol levels decreased. From December, the sucrose level markedly decreased in the bud.
Fig. 3. Seasonal variations of soluble sugars levels in the bud (}) and in the stem (&) and starch levels in the bud (^) and in the stem (&).
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Fig. 4. Soluble sugar contents in the bud (~) and in the stem (&).
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Fig. 4. (Continued )
4. Discussion In isolated tissues, sucrose absorption was higher than that of sorbitol. These results could explain, at least partially, the results of Moing et al. (1994) who observed that sucrose concentration was higher than that of sorbitol in vegetative buds and pieces of bark in prune trees. Similar observations were made on young leaves of peach trees (GaudilleÁre et al., 1989). In mature leaves, sucrose is used to synthesize sorbitol, increasing its concentration (Merlo and Passera, 1991). Sucrose and sorbitol absorption potentials were important in October in the stem and the bud. This situation proceeded just as temperature fell and could be related to a mechanism of freezing tolerance. During cold acclimation, some authors have noted an increase of intracellular osmotic potential due to an
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Fig. 5. General scheme of possible sugar metabolism in buds during dormancy (from October to December) and its release (from January to February) (a) and a representation of the flux orientation of sugars, for both periods, in the single node cutting (b).
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accumulation of soluble sugars and amino acids. Sucrose and raffinose could exert a cold protection (Koster and Lynch, 1992). Now, sucrose and stachyose raffinose levels were particularly increased from October in the bud and reached a maximum in December (for sucrose) and in January (for stachyose raffinose). The accumulation of soluble sugars, particularly polyols and sucrose (Young, 1969; Williams et al., 1990; Layne and Ward, 1978), is important for the freezing protection of cells by their ability to supply energy (Hansen and Grauslund, 1973), their osmotic effects (Sakai, 1960) and the protection of proteins and membranes (Young, 1969; Steponkus et al., 1977). The noted increase of total and active absorption can be explained by structural modifications of the plasma membrane, as previously shown in peach trees (Portrat et al., 1995; Wisniewski and Ashworth, 1986). At the same time, we observed in the bud, an increase of sucrose level linked to a decrease of glucose and fructose levels. The latter are probably used for sucrose synthesis. Fig. 5(a) represents the possible sugar metabolism occurring in buds during dormancy (from October to December) and its release (from January to February), integrating the data presented. During dormancy, both sorbitol and starch could be hydrolysed to fructose and glucose, respectively, leading to sucrose synthesis. This agreed with the flux orientation of sugars observed during dormancy (Fig. 5(b)): the fall of total and active absorption observed during bud dormancy, was concomitant with starch hydrolysis. In December, the bud, unable to attract osmotic and energetic molecules, used its reserves (starch) to synthesize osmotic molecules such as sucrose, stachyose and raffinose. During dormancy release (from January), sucrose was used in buds (Fig. 5(a)) to synthesize the sorbitol and stachyose raffinose that are required for budburst. It is noteworthy that although the bud reacquired its active absorption potential for exogeneous sucrose (Fig. 5(b)), sucrose levels nevertheless decreased. At this time, the rate of supply by the bud's underlying tissues was insufficient to restore a high sucrose level. The accumulation of soluble sugars noted during dormancy release could partially allow the buds growth. Finally, two periods can be distinguished (Fig. 5(b)) concerning the flux orientation and the use of sugar within the single node cutting. During dormancy, the bud, exhibited a low absorption potential for nutrients, it hydrolysed the starch reserves and increased its freezing protection by sucrose synthesis. During dormancy release, the bud exhibited an important sink strength by active transport and was able to accumulate carbon reserves (sorbitol, stachyose raffinose and starch) which were used for growth metabolism, inducing budburst. References GaudilleÁre, J.P., Moing, A., Lamant, A., Brion, M., Schaeffer, J., 1989. Compartmentation of storage compounds in peach leaves. Ann. Sci. For. 46, 828s±831s.
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GaudilleÁre, J.P., Moing, A., Carbonne, F., 1992. Vigour and non-structural carbohydrates in young prune trees. Scientia Hort. 51, 197±211. Gendraud, M., PeÂtel, G., 1990. Modifications in intercellular communications, cellular characteristics and change in morphogenetic potentialities of Jerusalem artichoke tubers (Helianthus tuberosus L.). In: Millet, B., Greppin, H. (Eds.), Intra- and Intercellular Communications in Higher Plants: Reception, Transmission, Storage and Expression of Messages. INRA editions, Paris, pp. 170±175. Hansen, P., Grauslund, J., 1973. 14 C-studies on apple trees. VII. The seasonal variation and nature of reserves. Physiol. Plant 28, 24±32. Koster, K.L., Lynch, D.V., 1992. Solute accumulation and compartmentation during the cold acclimation of Puma Rye. Plant Physiol. 98, 108±113. Kunst, A., Draeger, B., Ziegenhorn, J., 1984. Carbohydrates, U.V. method with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer, H.U. (Ed.), Methods in Enzymate Analysis, 3rd ed. VI, Metabolites 1. Verlag Chemie, Basel, pp. 163±172. Lang, G.A., Early, J.D., Martin, G.C., Darnell, R.L., 1987. Endo-, para-, and ecodormancy: physiological terminology and classification for dormancy research. Hortsc. 22, 371±377. Layne, R.E.C., Ward, G.M., 1978. Rootstock and seasonal influences on carbohydrate levels and cold hardiness of Redhaven peach. J. Amer. Soc. Hort. Sci. 103. Loescher, W.H., 1987. Physiology and metabolism of sugar alcohols in higher plants. Physiol. Plant. 70, 553±557. Marquat, C., PeÂtel, G., Gendraud, M., 1997. Biochemical characterization of saccharose and sorbitol transporters from plasmalemma membrane vesicles of peach-tree leaves. Biol. Plant. 39, 369±378. M'Batchi, B., Delrot, S., 1984. Parachloromercuribenzenesulfonic acid. Plant Physiol. 75, 154±160. Merlo, L., Passera, C., 1991. Changes in carbohydrate and enzyme levels during development of leaves of Prunus persica, a sorbitol synthesizing species. Physiol. Plant. 83, 621±626. Moing, A., Lafargue, B., Lespinasse, J.-M., GaudilleÁre, J.-P., 1994. Non-structural carbohydrates in flower buds and vegetative buds in prune trees. Acta Hort. 359, 287±294. Portrat, K., Mathieu, C., Motta, C., PeÂtel, G., 1995. Changes in plasma membrane properties of peach-tree buds and stands during dormancy. J. Plant Physiol. 147, 346±350. Sakai, A., 1960. Relation of sugar content to frost hardiness in plants. Nature 185, 698±699. Sakr, S., Lemoine, R., Gaillard, C., Delrot, S., 1993. Effect of cutting on solute uptake by plasma membrane vesicles from sugar beet (Beta Vulgaris L.) leaves. Plant Physiol. 103, 49±58. Steponkus, P.L., Garber, M.P., Myers, S.P., Lineberger, R.D., 1977. Effects of cold acclimation and freezing on structure and function of chloroplast thylakoids. Cryobiology 14, 303±321. Williams, L.E., Nelson, S.J., Hall, J.L., 1990. Characterization of solute transport in plasma membrane vesicles isolated from cotyledons of Ricinus communis L. II ± Evidence for a protoncoupled mechanism for sucrose and amino acid uptake. Planta 182, 540±545. Wisniewski, M., Ashworth, E.N., 1986. A comparison of seasonal ultrastructural changes in stem tissues of peach (Prunus persica) that exhibit contrasting mechanisms of cold hardiness. Bot. Gaz. 147, 407±417. Young, R., 1969. Cold hardening in citrus seedlings as related to artificial hardening conditions. J. Amer. Soc. Hort. Sci. 94, 612±614.
Dormancy in vegetative buds of peach: relation between carbohydrate absorption potentials and carbohydrate concentration in the bud during dormancy and its release Christel Marquata, Marc Vandammeb, Michel Gendrauda, Gilles PeÂtela,* a Unite AssocieÂe Bioclimatologie - P.I.A.F. (INRA - Universite Blaise Pascal), Physiologie et GeÂneÂtique veÂgeÂtales, 24 avenue des Landais, 63177, AubieÂre CedeÂx, France b INRA-Station de Bioclimatologie, Domaine de Crouelle, 63069, Clermont-Ferrand Cedex, France
Accepted 31 July 1998
Abstract The absorption of sucrose and sorbitol by the bud and the stem of Prunus persica was evaluated during the rest period. The utilization of p-chloromercuribenzenesulfonic acid (PCMBS), an inhibitor of sucrose transporter, allowed estimation of the active absorption of sucrose in different isolated organs (bud, bud's underlying tissues and stem). It was assumed that the sink capacities of tissues depended on their potential to absorb carbohydrates by active transport. Endogenous carbohydrate levels in the tissues were also measured. In the bud, the qualitative and quantitative distribution of carbohydrates could be related to the absorption potential of the bud. During dormancy, the bud exhibited a low absorption potential and increased its sucrose concentration by starch hydrolysis. Soluble sugars accumulated during winter. During dormancy release, the bud was able to absorb carbohydrates allowing carbon storage. The data presented led to the proposal of a scheme of events occurring during bud dormancy and its release. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Vegetative bud; Absorption potentials; Sucrose; Sorbitol; Carbon storage
* Corresponding author. Fax: +33-473-407934. 0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 8 ) 0 0 2 0 3 - 9
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1. Introduction Dormancy can have three causes according to Lang et al. (1987): (i) an endogeneous signal within the affected structure (endodormancy); (ii) a biochemical signal originating in a structure other than the affected structure (paradormancy); (iii) environmental factors which affect over all plant metabolism (ecodormancy). The factors which control the induction and duration of dormancy have not been elucidated until now. In Prunus persica (L.) Batsch, the induction of vegetative bud dormancy takes place during late August and budburst occurs at the end of February. The state of rest could be linked, at least partially, to trophic correlations between the bud's underlying tissues and the bud itself, thus regulating its growth (paradormancy). In peach, during the bud's rest period, absorption of nutrients by the bud's underlying tissue and the stem could lead to the bud's deprivation and reduce its ability for growth (Gendraud and PeÂtel, 1990). Our work aims to identify (i) the absorption potential of the bud and the stem, in Prunus persica, during the rest period and during dormancy release; (ii) the variation in the major soluble sugars (sucrose, fructose, glucose, sorbitol and raffinose stachyose) and starch in the stem and the bud. Active absorption of sucrose was measured by the use of PCMBS (p-chloromercuribenzenesulfonic acid) to inhibit the sucrose transporter (M'Batchi and Delrot, 1984; Sakr et al., 1993), while allowing simple diffusion. The effect of this inhibitor was previously evaluated in situ on the organs of single node cuttings (Marquat et al., 1997). These two methods allowed the bud's dormancy to be related to its sugar absorption potential by active transport and to the level and the use of its carbohydrate content. 2. Material and methods 2.1. Plant material Current year twigs were sampled from 10 year-old peach trees (Prunus persica L.). Budburst occurred at the end of February or at the beginning of March. Only the axillary and isolated vegetative buds were used. For the estimation of absorption potentials, 3 samples were used for each organ (scaled bud and stem) from October to January and 5 different measurements were made on each sample. For the measurement of soluble sugars and starch levels, 50 scaled buds and stem pieces were collected every month from October to January. They were stored at ÿ808C, then lyophilized. Dried samples were ground to a fine powder and weighed.
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2.2. Determination of solute uptake by different tissues of peach Each isolated organ (scaled bud, stem discs) was weighed and immersed, for 2 h, in 400 ml of a medium containing 300 mM mannitol, 20 mM Mes-KOH (pH 6.5), 0.5 mM CaCl2, 0.25 mM MgCl2, either in the presence or in the absence (control) of 1 mM PCMBS. Mannitol concentration was changed to maintain a constant osmolarity. PCMBS was used in order to evaluate `active' absorption. Samples were rinsed with a control medium for 30 min and then immersed in a control medium labelled with 300 mM [3 H] mannitol (specific activity 0.97 TBq mmolÿ1; Sigma, USA). Uptake was initiated by adding 1 mM [14 C] sucrose (specific activity 23 GBq mmolÿ1; Amersham, UK) or 1 mM [14 C] sorbitol (specific activity 12 GBq mmolÿ1; Amersham, UK) and run at 238C for 3 h. Shorter times (1 h, 2 h) were tested but were insufficient, as 3 h corresponded to the time necessary to obtain the diffusion equilibrium. Samples incubated with [3 H] mannitol allowed us to determine the free space. As mannitol does not diffuse into the intracellular medium and is not transported, it allowed the incorporation of sucrose into the intracellular medium to be evaluated. At diffusion equilibrium, the 14 C=3 H ratio was compared with the initial 14 C=3 H ratio in the medium (0.5). After this period, each tissue was rinsed with the same control medium, dried, and homogenized using a mortar. Labelled sugars were extracted from the homogenates in 80% ethanol for 2 min at 1008C. Radioactivity was measured by scintillation counting in the presence of 5 ml liquid scintillation cocktail. This experiment allowed evaluation of the sucrose transporter's specificity for sucrose and the protecting effect of the sucrose on PCMBS inhibition. 2.3. Sugar extraction We followed the procedure described by GaudilleÁre et al. (1992). Seven mg of dried material (scaled buds 2 mm in length and stem discs 4 mm in diameter and 1 mm thick) were used for analysis. Soluble sugars were extracted with boiling 80% ethanol for 30 min. Extracts were centrifuged at 15 000g for 10 min in order to remove insolubles (starch). This operation was repeated twice. The alcohol fraction containing the soluble sugars was evaporated to dryness under vacuum. The pellet was solubilized in 500 ml of ultra-pure water. Soluble sugars were purified using anion- (DOWEX 50 W 50*8 200±400 mesh form-H) and cation-exchange (BIORAD AGI*8 100±200 mesh form HCO3) resins to remove organic phosphate, amino acids and organic acids. The purified extracts were dried for 12 h under vacuum and were then resuspended in 500 ml of H2O.
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2.4. Carbohydrate analysis Soluble carbohydrates were determined by HPLC. One hundred ml of extract was filtered on Millipore filter (0.45 mm) adapted on a syringe. Twenty ml of each extract was separated in an HPLC system consisting of a pre-column (BIORAD Carbo P Micro Guard 30 4.6 mm) and a column (BIORAd Aminex HPX 87 P 300 7.8 mm). The carbohydrates were quantified from peak area calculations related to regression curves of standards. Sorbitol content was measured by an enzymatic method (Boehringer-Mannheim) based on the following enzymatic reactions (Kunst et al., 1984 and Loescher, 1987): sorbitol NAD
!
sorbitol dehydrogenase
fructose NADH H
(1)
To avoid reversion of this first reaction, the (NADH H) obtained is transformed to NAD according to Eq. (2). This also ensured the complete hydrolysis of sorbitol: NADH H pyruvate
!
lactate dehydrogenase
lactate NAD
(2)
The fructose obtained from Eq. (1) is then transformed to glucose-6-phosphate, as follows: fructose ATP ! fructose-6-phosphate
(3)
fructose-6-phosphate ! glucose-6-phosphate
(4)
The last reaction, catalysed by the glucose-6-phosphate dehydrogenase, leads to the formation of NADPH H: glucose-6-phosphate NADP ! gluconate-6-phosphate NADPH H (5) (NADPH H) formation was quantified at 340 nm and corresponded to the quantity of oxidised sorbitol (Eq. (1)). The pellets were dried under vacuum for 30 min, solubilized in 1 ml NaOH 0.02 N and placed at 908C for 30 min in order to remove cell walls and to solubilize the starch. 50 ml of the suspension were incubated with 100 ml of citrate buffer (pH 4.2) containing 15 units of amyloglucosidase (Sigma) for 30 min at 508C and centrifuged at 15 000g. The glucose released was quantified by measuring the formation of NADPH H at 340 nm (Kunst et al., 1984), according to the following reaction: glucose ATP ! glucose-6-phosphate The glucose-6-phosphate was then transformed to gluconate-6-phosphate, according to Eq. (5), inducing the formation of NADPH H.
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3. Results 3.1. Sucrose absorption Total sucrose absorption increased between September and October in both the stem and the bud. In the bud, total absorption decreased in December (Fig. 1(a)). In the stem, total sucrose absorption decreased a little from November (Fig. 1(b)). Active absorption decreased from October in both tissues. In December, both
Fig. 1. Changes in active (&) and total (&) absorption of sucrose in the bud (a) and in the stem (b). Each experiment was conducted on 3 different samples. Data are means S.E. of 5 measurements from 3 different samplings.
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total and active uptake were higher in the stem than in the bud. The stem values did not differ from the values found in October. In January, the ability of the bud to accumulate sucrose increased markedly and active absorption reached a maximum. In the stem, active absorption was also important in January. Globally, an enhancement of active sucrose absorption was observed in January. 3.2. Sorbitol absorption Sorbitol absorption was not inhibited by PCMBS. So, study of active absorption of sorbitol in the different organs was not possible (Marquat et al., 1997). Variation in sorbitol absorption potential was less for sucrose (Fig. 2). In the stem, sorbitol absorption reached a minimum in November and a maximum in December. In the bud, absorption was low in November and December, then increased in January. In December, the stem exhibited an important sink strength for sorbitol. In January, absorption potentials were comparable in the stem and in the bud. As no experiments were previously monitored using vegetative buds of peach, soaking scaled buds in sugar solution may not constitute a valid simulation of the actual transport of sugars from the stem into buds. So, an alternative method was tested in which the cut end of a single node cutting was placed on the sugar solution. Under these conditions, total absorption of sucrose and sorbitol in the buds was measured. Similar results were observed using both methods. For instance, in October, total absorption of sucrose was 1.4 nmol/mg FW using the single node cutting method and 1.3 nmol/mg FW using the soaked scaled buds
Fig. 2. Changes in total absorption of sorbitol in the bud (&) and in the stem (&). Each experiment was conducted on 3 different samples. Data are means S.E. of 5 measurements from 3 different samplings.
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method. The same observation was made concerning sorbitol absorption. Unfortunately, the single node cutting method could not be used to measure the active absorption of sugars, requiring the use of PCMBS. Experiments showed that PCMBS action, in this condition, was not reproducible enough to obtain useful data. Consequently, the soaked scaled buds method, reflecting the actual absorption of sugars from stem to buds through vascular bundles was chosen. 3.3. Carbohydrate contents of buds and stem Whatever the month, the buds had a higher level of total soluble sugars (three times that of the stem) (Fig. 3). In the stem, 50% of starch was hydrolysed between October and December (Fig. 3). From December, starch reserves increased (5 mg/g DW) and soluble sugars decreased (9 mg/g DW). In the bud, starch was hydrolysed from October until December. From December, starch level increased in the bud and soluble sugars level decreased by a similar amount (30 mg/g DW). In November, stachyose and raffinose were detected in both the stem and the bud (Fig. 4(a)). In the stem, their concentration progressively decreased until February. In the bud, stachyose raffinose levels increased to a high value in January. In the stem, sucrose and sorbitol levels were constant (Fig. 4(b) and (c)). Fructose and glucose levels increased until December, then decreased until budburst. In the bud, sucrose and sorbitol represented 78.5% of the total soluble sugars. Between October and December, the sucrose level increased and glucose (Fig. 4(d)), fructose (Fig. 4(e)) and sorbitol levels decreased. From December, the sucrose level markedly decreased in the bud.
Fig. 3. Seasonal variations of soluble sugars levels in the bud (}) and in the stem (&) and starch levels in the bud (^) and in the stem (&).
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Fig. 4. Soluble sugar contents in the bud (~) and in the stem (&).
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Fig. 4. (Continued )
4. Discussion In isolated tissues, sucrose absorption was higher than that of sorbitol. These results could explain, at least partially, the results of Moing et al. (1994) who observed that sucrose concentration was higher than that of sorbitol in vegetative buds and pieces of bark in prune trees. Similar observations were made on young leaves of peach trees (GaudilleÁre et al., 1989). In mature leaves, sucrose is used to synthesize sorbitol, increasing its concentration (Merlo and Passera, 1991). Sucrose and sorbitol absorption potentials were important in October in the stem and the bud. This situation proceeded just as temperature fell and could be related to a mechanism of freezing tolerance. During cold acclimation, some authors have noted an increase of intracellular osmotic potential due to an
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Fig. 5. General scheme of possible sugar metabolism in buds during dormancy (from October to December) and its release (from January to February) (a) and a representation of the flux orientation of sugars, for both periods, in the single node cutting (b).
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accumulation of soluble sugars and amino acids. Sucrose and raffinose could exert a cold protection (Koster and Lynch, 1992). Now, sucrose and stachyose raffinose levels were particularly increased from October in the bud and reached a maximum in December (for sucrose) and in January (for stachyose raffinose). The accumulation of soluble sugars, particularly polyols and sucrose (Young, 1969; Williams et al., 1990; Layne and Ward, 1978), is important for the freezing protection of cells by their ability to supply energy (Hansen and Grauslund, 1973), their osmotic effects (Sakai, 1960) and the protection of proteins and membranes (Young, 1969; Steponkus et al., 1977). The noted increase of total and active absorption can be explained by structural modifications of the plasma membrane, as previously shown in peach trees (Portrat et al., 1995; Wisniewski and Ashworth, 1986). At the same time, we observed in the bud, an increase of sucrose level linked to a decrease of glucose and fructose levels. The latter are probably used for sucrose synthesis. Fig. 5(a) represents the possible sugar metabolism occurring in buds during dormancy (from October to December) and its release (from January to February), integrating the data presented. During dormancy, both sorbitol and starch could be hydrolysed to fructose and glucose, respectively, leading to sucrose synthesis. This agreed with the flux orientation of sugars observed during dormancy (Fig. 5(b)): the fall of total and active absorption observed during bud dormancy, was concomitant with starch hydrolysis. In December, the bud, unable to attract osmotic and energetic molecules, used its reserves (starch) to synthesize osmotic molecules such as sucrose, stachyose and raffinose. During dormancy release (from January), sucrose was used in buds (Fig. 5(a)) to synthesize the sorbitol and stachyose raffinose that are required for budburst. It is noteworthy that although the bud reacquired its active absorption potential for exogeneous sucrose (Fig. 5(b)), sucrose levels nevertheless decreased. At this time, the rate of supply by the bud's underlying tissues was insufficient to restore a high sucrose level. The accumulation of soluble sugars noted during dormancy release could partially allow the buds growth. Finally, two periods can be distinguished (Fig. 5(b)) concerning the flux orientation and the use of sugar within the single node cutting. During dormancy, the bud, exhibited a low absorption potential for nutrients, it hydrolysed the starch reserves and increased its freezing protection by sucrose synthesis. During dormancy release, the bud exhibited an important sink strength by active transport and was able to accumulate carbon reserves (sorbitol, stachyose raffinose and starch) which were used for growth metabolism, inducing budburst. References GaudilleÁre, J.P., Moing, A., Lamant, A., Brion, M., Schaeffer, J., 1989. Compartmentation of storage compounds in peach leaves. Ann. Sci. For. 46, 828s±831s.
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