winter variation in xylem sap ph of walnut trees.full

Summary We studied seasonal variation in xylem sap pH ofJuglans regia L. Our main objectives were to (1) test the effectof temperature on seasonal changes in xylem sap pH and (2)study the involvement of plasma membrane H+-ATPase of ves-sel-associated cells in the control of sap pH. For this purpose,orchard-grown trees were compared with trees grown in aheated (≥ 15 °C) greenhouse. During autumn, sap pH was notdirectly influenced by temperature. A seasonal change inH+-ATPase activity resulting from seasonal variation in theamount of protein was measured in orchard-grown trees,whereas no significant seasonal changes were recorded ingreenhouse-grown trees. Our data suggest that H+-ATPasedoes not regulate xylem sap pH directly by donating protons tothe xylem, but by facilitating secondary active H+/sugartransport, among other mechanisms.

Keywords: Juglans regia, temperature, winter functioning.

Introduction

Xylem sap is generally acidic, ranging between pH 5 and 6(Bollard 1960). However, there is evidence that xylem sap pHcan vary seasonally, becoming neutral or even alkaline duringsome periods of the year. In Acer pseudoplatanus L., xylemsap pH is close to 6.9 in February (Essiamah 1980) and canreach 7.5 in Betula pendula Roth by the end of winter (Sauterand Ambrosius 1986). In species such as Actinidia chinen-sis L. (Ferguson et al. 1983), Betula pendula (Sauter andAmbrosuis 1986) and Populus × canadensis “robusta”(Sauter 1988), pH values are close to neutral in winter andacidic at the beginning of spring. The acidification mechanismis not understood, but the cell type best localized to play a ma-jor role in the control of xylem sap pH is the vessel-associatedcell (VAC) (Sauter 1988, De Boer and Volkov 2003). Alves etal. (2001) reported that the structural characteristics of VACsin walnut are similar to those described for VACs in other spe-cies (Läuchli et al. 1974, Van Bel and van der Schoot 1988).

Vessel-associated cells are small, have a high nucleoplasmicratio and surround the xylem vessels. They are characterizedby a dense cytoplasm that contains a voluminous nucleus,small and numerous vacuoles and many organelles and inclu-sions. Nearly half of the cell surface area in contact with thevessel has large pits. There are many symplasmic connectionsbetween VACs and rays and axial parenchyma cells (Lachaudand Maurousset 1996, Alves et al. 2001).

In Robinia pseudoacacia L., seasonal variations in the pH ofsolution perfused through the vessels, and the effects on thispH value of the protonophore carbonyl cyanide-m-chloro-phenylhydrazone (CCCP) and of fusicoccin (FC), a specificactivating agent of plasma membrane H+-ATPase (Fromard etal. 1995), indicate that some living cells in wood tissue are in-volved in the control of vascular sap pH and that this controlfluctuates seasonally. These patterns are similar to the pH vari-ations occurring in the vascular sap of various woody speciesthroughout the year and are characterized by a pronouncedacidification in early spring. Essiamah (1980) suggested thatthe acidification was a result of the arrival of more acidic sapfrom the base of the tree. However, the observed acidificationof tracheal sap in spring could also be explained, at least inpart, by a proton-coupled sucrose efflux into the sap, as re-ported by Humphreys and Smith (1980) in Zea mays L.scutellum slices. It has also been suggested that spring acidifi-cation of the vascular sap is closely related to plasma mem-brane H+-ATPase activity (EC 3.6.1.35) of VACs (Fromard etal. 1995). Moreover, in spring, immunolabeling of plasmamembrane H+-ATPase in VACs has been observed, suggestingthat VACs have enough of this enzyme to control the pH ofvascular sap.

Seasonal changes in plasma membrane H+-ATPase havebeen studied during cambial growth in Populus nigra L. andPopulus trichocarpa Torr. & A. Gray (Arend et al. 2002). Dur-ing autumn and winter dormancy, only a slight immuno-reactivity against the plasma membrane H+-ATPase was foundin cross sections and tissue homogenates. In contrast, in spring

Tree Physiology 24, 99–105© 2004 Heron Publishing—Victoria, Canada

Winter variation in xylem sap pH of walnut trees: involvement ofplasma membrane H+-ATPase of vessel-associated cells

GEORGES ALVES,1 THIERRY AMEGLIO,2 AGNES GUILLIOT,1 PIERRETTEFLEURAT-LESSARD,3 ANDRÉ LACOINTE,2 SOULAÏMAN SAKR,1 GILLES PETEL1 andJEAN-LOUIS JULIEN1,4

1 U.M.R. PIAF, site des Cézeaux, Université Blaise Pascal, 24 avenue des Landais, 63177 Aubière cedex, France2 U.M.R. PIAF, site INRA de Crouelle, 234 avenue du Brézet, 63039 Clermont-Ferrand cedex 2, France3 U.M.R. CNRS 6161, Bâtiment Botanique, 40 avenue Recteur Pineau, 86022 Poitiers, France4 Author to whom correspondence should be addressed ([email protected])

Received November 26, 2002; accepted June 14, 2003; published online December 1, 2003

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during cambial growth, strong immunoreactivity was ob-served in cambial cells and expanding xylem cells. It seemsthat this H+-ATPase generates an H+ gradient that can drive theuptake of K+ and nutrients into cambial and expanding xylemcells. It was also demonstrated that plasma membraneH+-ATPase activity varied considerably during the growingseason (Iivonen and Vapaavuori 2002).

The objective of our study was to investigate the possiblerole of plasma membrane H+-ATPase of VACs in the control ofxylem sap in walnut trees (Juglans regia L.). The activity ofplasma membrane H+-ATPase was studied by a perfusiontechnique, bioluminescence activity assays and Western blotanalyses. The study was performed in winter and early springin walnut trees growing in an orchard. Walnut trees kept in aheated greenhouse (≥ 15 °C) served as a non-hardened refer-ence to characterize the effect of low temperature on thephysiological state of the xylem.

Materials and methods

Location, climatic data, plant material and treatments

Data were gathered for five winter seasons (1994–1998 and1999–2000) at the INRA PIAF located near Clermont-Ferrand, in south-central France. Twenty walnut trees (Juglansregia cv. Franquette) were grown in orchards and were13 years old in 1994. Daily maximum and minimum air tem-peratures were measured at the weather station at Aulnat(Météo France), which is located less than 1 km away from theINRA.

Thermal conditioning experiments were performed on con-tainer-grown trees. In 1997–1998, 1998–1999 and 1999–2000, ten 3-year-old walnut trees in individual 33-l pots weregrown outdoors until late summer. The pots contained a 1:2(v/v) mixture of peat and clay soil that was well-drained anddrip-irrigated to field capacity each day during the growingseason. In late September, the trees were transferred to aheated greenhouse (temperature between 15 and 25 °C) to en-sure that they did not harden during the winter. Because thegreenhouse trees were potted and the outdoor trees were in anorchard, we irrigated the container-grown trees to field capac-ity each week to minimize water stress.

Biological measurements

Measurements were performed on excised, 1-year-old twigssampled from orchard- and container-grown trees. Each twigwas at least 70 cm long. After removing the uppermost 10-cmapical part, the sub-adjacent 30-cm-long stem section wasused for the different experiments. It was verified that, whencomparing xylem sap pH values of different samples, be-tween-tree variability was not significantly different fromwithin-tree variability.

Sap extraction

Xylem sap was extracted from stem segments according to themethod of Bollard (1953) (see Schurr (1998) for a review).Sap extraction was carried out 1 h after twig excision at the

same time every day (0900 h). Around 10 cm of bark from theapical part of the stem was removed to avoid contaminationwith phloem sap. Stems were placed in a vacuum extractionsystem allowing simultaneous extraction from several stemsby applying 0.1 MPa suction. Sap samples were collected inglass tubes placed on ice. Sap pH was measured directly fol-lowing extraction with a microelectrode (Model N96-3621Broadley-James, Irvine, CA) During winters 1994 –1995 and1995–1996, sap pH was determined on two branches of eachof four orchard-grown trees (n = 8 measurements per date). Inwinters 1996–1997, 1997–1998 and 1999–2000, measure-ments were performed on each date on three branches fromeach of three trees (n = 9 measurements per date). During win-ter 1999–2000, sap pH was determined on three branches ofeach of two greenhouse-grown trees (n = 6 measurements perdate). Means with standard errors are presented.

Perfusion technique and pH measurements

Twelve-cm-long segments were collected from 1-year-oldtwigs after removing the apical 10 cm. Bark was removedfrom both ends of each segment and the ends were coveredwith paraffin film to prevent dehydration and contaminationwith phloem constituents. Three ml of unbuffered standard so-lution (0.1 mM KCl, 0.1 mM NaCl and 0.1 mM CaCl2 adjustedto pH 6.0 with 0.01 M HCl or NaOH) was passed through thestem segments by applying a gentle pressure (0.01 MPa) withcompressed air until a steady-state pH value, called the equi-librium pH, was obtained. The perfusate was unbuffered sothat the VACs would have maximal influence over pH. The ef-fects of the protonophore CCCP (10 µM) and the specific acti-vating agent of the plasma membrane H+-ATPase, FC (10 µM)were investigated by the perfusion technique described byAlves et al. (2001). The ∆pH is the difference between the pHmeasured in the presence of the effector and the equilibriumpH.

The pH of the perfusate was measured continuously with amicroelectrode (Type mini combo pH750, World Precision In-struments, Sarasota, FL) inserted into the droplet of theperfused solution.

Isolation of plasma membrane fraction

The plasma membrane fraction was isolated by a two-phaseaqueous partitioning technique as described by Alves et al.(2001). All samples were kept on ice throughout the proce-dure. Briefly, 20 g of xylem (separated from bark, phloem andcambium) from 1-year-old twigs (three samples of independ-ent stem and independent tree were used for each measure-ment) was ground in 100 ml of 50 mM HEPES-KOH (pH 7.5)with 10% (w/v) polyvinylpolypyrrolidine (PVPP), 0.5 M su-crose, 10 mM ascorbic acid, 3.6 mM cysteine, 0.5 mM DTTand 1 mM phenylmethanesulfonyl fluoride. The slurry was fil-tered through four layers of cheesecloth and centrifuged for15 min at 10,000 g. The supernatant was centrifuged for45 min at 73,000 g. The microsomal fraction was resuspendedin 1 ml of phosphate buffer (5 mM K2HPO4, adjusted to pH 7.8with KH2PO4) containing 0.3 M sucrose and 3 mM KCl. One

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gram of resuspended pellet was added to 14 g of phase mixturewith a final composition of 6% (w/v) Dextran, 6% (w/v) poly-ethylene glycol (PEG), 1 mM sucrose, 1 mM KCl and 5 mMphosphate buffer, pH 7.8. After mixing, the system was centri-fuged for 5 min at 3000 g. The upper phase was separated, di-luted with one volume of Tris buffer (adjusted to pH 6.5 withMES) containing 0.5 M sucrose and 0.5 mM DTT, and centri-fuged for 1 h at 100,000 g. The final pellets, corresponding tothe plasma membrane-enriched fractions, were taken up inTris buffer and stored at –80 °C.

Bioluminescence and protein assays

Plasma membrane H+-ATPase activity was measured in 10 µgof protein by bioluminescence as described by Alves et al.(2001). We used 0.015% (w/v) Brij 58 detergent to obtain vesi-cles of uniform sidedness. Plasma membrane H+-ATPase ac-tivity was measured at room temperature in 500 µl of 25 mMTris-MES buffer, pH 6.5, with 4 mM MgSO4 and 1 mM ATP.Activity was assayed in the presence of 1 mM NaN3, 50 mMNaNO3, 0.1 mM Na2MoO4, and in the presence or absence of500 µM vanadate or 10 µM FC. After 10 min, the ATP remain-ing was quantified with the Luciferine-luciferase reagent kit(ATP bioluminescence assay kit CLS II, Roche Diagnostics,Mannheim, Germany). Plasma membrane proteins were quan-tified according to Bradford (1976), with bovine serumalbumin as a standard.

SDS-PAGE and Western blot analyses

A 15-µg sample of plasma membrane proteins was subjectedto SDS-PAGE (polyacrylamide gel electrophoresis) accordingto Laemmli (1970). The gel system consisted of a 5% stackinggel and a 10% resolving gel. Electrophoresis was carried out ata constant voltage of 150 V for about 3 h.

After gel electrophoresis, polypeptides were transferredelectrophoretically to a nitrocellulose membrane (TransferMembrane, BioTraceTM NT, Pall, New York, NY). The trans-fer buffer contained 25 mM Tris, 193 mM glycin, 0.1% (w/v)SDS and 20% (v/v) methanol. The transfer was performed at aconstant current of 1 A for 1 h at room temperature.

Tris-buffered saline was used as the basic medium forimmunoblotting. Teleostan gelatin (Sigma, St. Louis, MO) 3%(w/v) and Tween 20 (0.75 ml l–1) were used in the blocking ofnitrocellulose filters. A rabbit immunopurified antiserumraised against a peptide of the central domain of the plasmamembrane H+-ATPase (peptide CDPKEARAGIREVHF) wasdiluted 1/2000, and the second antibody, conjugated to alka-line phosphatase, was diluted 1/3000 (monoclonal anti-rabbitIgG alkaline phosphatase conjugate, immunoglobulin fractionof mouse ascites fluid, clone RG96 from Sigma). The alkalinephosphatase substrates were nitroblue tetrazolium (NBT) and5-bromo-4-chloro-5-indolyl phosphatase (BCIP). The bandson the western were quantified by image analysis (Bio-1Dsoftware, Vilber Lourmat, Marne-la-vallée, France).

Results

pH measurements

Xylem sap pH measured during five winter seasons (1994 –1998 and 1999–2000) showed similar patterns; for simplifica-tion, therefore, the mean winter variation for the 5 years is pre-sented in Figure 1A. In orchard-grown walnut trees, xylem sappH was close to 7.0 at the beginning of autumn. It dropped rap-idly to 5.5 at the beginning of winter and then declined slowlyuntil the end of February to reach a minimum value of 5.1. Inspring, xylem sap pH increased and a transient alkalinizationwas observed in early April in all years investigated. A secondalkalinization period occurred in early May. Moreover, theseasonal variation in xylem sap pH appeared to parallel thevariation in mean air temperature measured in the orchard. Intrees kept in a greenhouse at 15 °C during winter, xylem sappH was close to 6.5 at the beginning of autumn; it decreasedrapidly to 5.4 in early winter, but thereafter no significantchange was observed until April (Figure 1B).

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WINTER VARIATION IN XYLEM SAP pH OF WALNUT TREES 101

Figure 1. (A) Autumn, winter and spring variations in air temperatureand vascular sap pH of orchard-grown walnut trees. Data shown aremeans ± SE for 5 years from October 1994 to May 2000. Sap pH wasmeasured twice monthly on 1-year-old twigs. (B) Autumn, winter andspring variations in vascular sap pH of walnut trees grown in an or-chard or kept in a greenhouse at ≥ 15 °C during the 1999–2000 period.Data shown are means ± SE of at least six measurements.



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We used a perfusion technique to determine the impact ofthe plasma membrane H+-ATPase of VACs on xylem sap pH.After perfusing 3 ml of standard solution, the pH of theperfused solution stabilized to a constant value (equilibriumpH), indicating that the stem segment was totally rinsed of itsoriginal sap. The equilibrium pH (mean value of the 3 years1998–2000) is presented in Figure 2A. In orchard-growntrees, the equilibrium pH of the solution perfused was 5.3 inFebruary and close to 5.7 in April. For the same periods, nosignificant change of the equilibrium pH value (5.7) was ob-served in trees kept in a greenhouse at 15 °C during winter.

After the pH stabilized to its equilibrium value, the per-fusing solution was supplemented with either CCCP or FC.The ∆pH after CCCP or FC addition is reported in Figure 2B.In orchard-grown trees, CCCP induced a higher alkalinizationof the perfusing liquid in April (0.28 pH unit) than in February(0.18 pH unit), revealing seasonal variations in the pH gradientbetween the cytoplasm of VACs and xylem sap. A similar pat-tern was observed in container-grown trees in the greenhouse.In orchard-grown trees, the FC treatment performed in Febru-

ary had no significant effect on the pH of the solution perfused,whereas FC induced acidification of about 0.33 pH units inApril, suggesting that VACs have enough plasma membraneH+-ATPase to control xylem sap pH. In greenhouse-growntrees, FC induced significant acidification of about 0.19 and0.10 in February and April, respectively.

Plasma membrane H+-ATPase activity and quantification

The purity of the plasma membrane-enriched fraction was es-timated with various ATPase effectors (data not shown). In xy-lem fractions, H+-ATPase activity was inhibited by about 80%by vanadate, whereas no significant inhibition by nitrate,molybdate or azide was recorded, indicating that the fractionswere free from mitochondria and thylakoid contamination.

In xylem tissue of orchard-grown trees, the specific activityof H+-ATPase increased from 6 to 30 nKat (mg protein)–1

from February to April (Figure 3A). Addition of FC increasedthe H+-ATPase activity by 9% relative to the control in Febru-ary and by 46% in April (Figure 3B). In trees kept in the green-house, H+-ATPase specific activity increased from 29 to35 nKat (mg protein)–1 from February to April (Figure 3A).Addition of FC increased H+-ATPase activity by 23% relative

102 ALVES ET AL.


Figure 2. Winter and spring variations in pH of the solution perfusedthrough 1-year-old stem segments harvested from walnut trees grownin an orchard or kept in a greenhouse at ≥ 15 °C. Experiments wereperformed in 1998, 1999 and 2000. (A) Seasonal variations in theequilibrium pH. Mean equilibrium pH values ± SE (n ≥ 30) are re-ported. (B) Effects of cyanide-m-chloro-phenylhydrazone (CCCP)(white bar) and fusicoccin (FC) (solid bar) on the pH of the perfusedsolution, after equilibrium. Mean ∆pH values ± SE (n ≥ 15) are re-ported.

Figure 3. (A) Plasma membrane H+-ATPase specific activity mea-sured in plasma membrane-enriched fraction obtained from 20 g(fresh weight) of xylem tissue in winter and spring of 1999 and 2000.Each value is the mean of five measurements ± SE. (B) Stimulation ofxylem plasma membrane H+-ATPase activity by fusicoccin (FC) in1999 and 2000. Each value is the mean of five measurements ± SE.



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to the control in February and by 31% in April (Figure 3B).Plasma membrane H+-ATPase antibody cross-reacted with

the plasma membrane vesicles that were isolated from walnutstem in the 100 kDa region (Figure 4). In February, the amountof plasma membrane H+-ATPase of xylem was higher in thetrees kept in the greenhouse than in the orchard-grown trees(Figure 4). In the orchard-grown trees, the amount of plasmamembrane H+-ATPase was higher in April than in February.The amount of plasma membrane H+-ATPase in the trees keptin the greenhouse was high and did not change between Febru-ary and April. Moreover, it seemed that, in April, the amountof plasma membrane H+-ATPase was similar in orchard- andgreenhouse-grown trees.

Discussion

Seasonal variation in xylem sap pH and the possible involve-ment of the plasma membrane H+-ATPase in the control ofvascular sap pH were investigated in walnut trees. In or-chard-grown trees (Figure 1A), xylem sap pH was close toneutral in October, then decreased rapidly to acidic values un-til February, and a transient alkalinization was systematically

observed at the beginning of April. These results suggest thatthe seasonal variation in xylem sap pH is correlated to temper-ature as reported by Fromard (1990) in Acer platanoides L. fortemperatures below zero. However, we observed (Figures 1Aand 1B) that xylem sap pH decreased rapidly to acidic valuesduring autumn in both orchard- and greenhouse-grown trees,indicating that, at least during this period, sap pH was not di-rectly influenced by temperature.

In contrast to walnut trees, sap pH in many woody species isclose to neutral in winter and becomes acidic (pH 5.5) at thebeginning of spring (Ferguson et al. 1983, Sauter andAmbrosius 1986, Sauter 1988, Fromard 1995). We used sev-eral methods to investigate the role of H+-ATPase in the regu-lation of vascular sap pH in walnut trees. The use of aperfusion technique allowed continuous monitoring of thevariation in pH of the perfused solution. In orchard-growntrees, the pH gradient revealed by use of CCCP and the ampli-tude of the acidification caused by FC showed seasonal varia-tions, with the responses being highest in April and lowest inFebruary (Figure 2B). This pattern in H+-ATPase activitycould be mainly explained by seasonal variation in the amountof protein. A similar seasonal variation in the amount of theplasma membrane H+-ATPase was recently reported duringcambial growth in Populus nigra and Populus trichocarpa(Arend et al. 2002). Although it is clear that VACs represent aminor component of the living cells of xylem in branches com-pared with other parenchyma cells, it has been demonstratedthat immunostaining of the plasma membrane H+-ATPase ismuch stronger in VACs than in other living cell types of thexylem (Fromard et al. 1995).

The equilibrium pH values (Figure 2A) of the perfused solu-tion showed similar variations to those measured directly insap collected by vacuum extraction (Figure 1). The differencebetween the pH of crude sap and the equilibrium pH of theperfused solution revealed short-term pH modifications of thestandard solution. The acidification of the perfused solution inFebruary in the orchard-grown trees (Figure 2A) confirmedthe capacity of the living cells of the xylem tissue to modifyvascular sap pH. In Robinia pseudoacacia, Fromard et al.(1995) proposed that the plasma membrane H+-ATPase ofVACs could control vascular sap pH and suggested that springacidification of the vascular sap was closely related to the ac-tivity of the enzyme. However, our observation that winteracidification occurred in the perfusate (Figure 2A) of the or-chard-grown trees when H+-ATPase activity was lowest (Fig-ure 3A) casts some doubt on the validity of this hypothesis forwalnut. Our observation is supported by the study of Gerendasand Schurr (1999) who observed that proton concentration it-self has little influence on xylem sap pH. Furthermore, wehave obtained circumstantial evidence for the existence of analternative non-H+-ATPase mechanism for the acidification ofxylem sap in walnut in winter.

As recommended by Gerendas and Schurr (1999), a detailedanalysis of the composition of the xylem sap is necessary toidentify which compound(s) among organic ions, inorganicions, amino acids and partial pressure in CO2 might be respon-sible for the winter acidification of xylem sap. As late as No-



Figure 4. Changes in amount of xylem plasma membrane H+-ATPasein walnut trees grown in an orchard or kept in a greenhouse in winterand spring of the 1999–2000 season. Upper panel: Western blot of theplasma membrane H+-ATPase (Mr ≈ 100 kDa) from 1-year-old stemsof walnut xylem tissue. Lanes 1 to 4 were loaded with 15 µg protein.Molecular mass markers (kDa) are indicated. Lower panel: Eachvalue represents the mean band intensity (± SE) of three replicates.



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vember, positive pressures were detected in intact plants butnot in excised segments (Ewers et al. 2001). These authorsconcluded that autumn pressures appeared to be of root originand demonstrated that they were associated with the uptake ofmineral nutrients from soil, especially nitrate. In February, thestem pressure appeared to be of stem origin, resulting in theaccumulation of sugars in the sap.

In walnut, xylem sugar accumulation is correlated with therepair of winter xylem embolism, resulting in xylem vesselconductivity restoration (Améglio et al. 1995, Améglio et al.2002). Xylem sugar accumulation results from the balance oftwo opposite movements: an efflux of sugars from paren-chyma cells into the xylem vessels (Sauter 1980) and an influxof sugars from the xylem sap into VACs (Sauter 1981). Currenthypotheses about the mechanism for both movements includefacilitated diffusion for the efflux (Sauter 1982) and anH+/sugar symport for the influx (Fromard 1990). This influxcould be dependent on plasma membrane H+-ATPase. In wal-nut, the efflux of sugar seems to drive local water movements(Lacointe et al. 1995). However, the mechanisms of the twofluxes remain hypothetical. It is possible that the sugar effluxoccurring in winter in walnut is responsible for sap acidifica-tion.

Moreover, the nonsignificant acidification of the perfusate,measured in April in the orchard-grown trees, and the acidifi-cation in both winter and spring in trees kept in the green-house, when H+-ATPase activity was high, confirms that thevascular sap pH does not depend directly on an increase in pro-tons being pumped into the xylem vessels.

In early spring, sap pH of the orchard-grown trees increasedtransiently (Figure 1A) when H+-ATPase activity was high(Figure 3A). Because our trees were well-watered, it seemsunlikely that this increase in sap pH occurred in response to adrought signal as previously reported in Commelina com-munis L. by Wilkinson and Davies (1997). We propose thatthis alkalinization is associated with secondary active H+-cou-pled cotransports. It is assumed that phloem is inactive duringwinter and that sugar mobilization at bud break occurs throughthe xylem pathway (Sauter and Ambrosius 1986, Lacointe etal. 2001). The increase in H+-ATPase activity at the beginningof spring could permit massive sugar uptake via an H+/sugarsymport in the VACs (Fromard 1990) leading to the alka-linization of the sap pH.

In the walnut trees kept in the greenhouse, no seasonal varia-tions in equilibrium pH were observed (Figure 2A). Further-more, neither the H+-ATPase activity (Figure 3A) nor theamount of protein (Figure 4) showed a significant seasonalvariation. Compared with the orchard-grown trees, these dataindicated that winter temperatures had an effect on H+-ATPaseactivity. In the trees kept in the greenhouse, the high ATPaseactivity measured throughout the winter (Figure 3) would beenergy-consuming, leading to the depletion of reserves thatcould account for the erratic bud break reported by Améglioand Cruiziat (1992). These results also indicate the need tostudy reserve mobilization in cold-deprived trees.

To summarize, we showed seasonal variation in xylem sappH of walnut trees and demonstrated that this pattern is not di-

rectly attributable to protons contributed by ATPase. Furtherstudies are necessary to elucidate the origin of the winter acidi-fication of xylem sap, the efflux mechanism and to confirm theinvolvement of a secondary active H+/sugar influx in springalkalinization.

Acknowledgments

We thank C. Bodet, S. Ploquin and P. Chaleil for their technical assis-tance. The skillful help of M. Bonhomme, N. Brunel and C. Beaudoinis gratefully acknowledged.

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