Tree Physiology 24, 99–105
© 2004 Heron Publishing—Victoria, Canada
Winter variation in xylem sap pH of walnut trees: involvement of
plasma membrane H+-ATPase of vessel-associated cells
GEORGES ALVES,1 THIERRY AMEGLIO,2 AGNES GUILLIOT,1 PIERRETTE
FLEURAT-LESSARD,3 ANDRÉ LACOINTE,2 SOULAÏMAN SAKR,1 GILLES PETEL1 and
JEAN-LOUIS JULIEN1,4
1
U.M.R. PIAF, site des Cézeaux, Université Blaise Pascal, 24 avenue des Landais, 63177 Aubière cedex, France
2
U.M.R. PIAF, site INRA de Crouelle, 234 avenue du Brézet, 63039 Clermont-Ferrand cedex 2, France
3
U.M.R. CNRS 6161, Bâtiment Botanique, 40 avenue Recteur Pineau, 86022 Poitiers, France
4
Author to whom correspondence should be addressed ([email protected])
Received November 26, 2002; accepted June 14, 2003; published online December 1, 2003
Summary We studied seasonal variation in xylem sap pH of
Juglans regia L. Our main objectives were to (1) test the effect
of temperature on seasonal changes in xylem sap pH and (2)
study the involvement of plasma membrane H+-ATPase of vessel-associated cells in the control of sap pH. For this purpose,
orchard-grown trees were compared with trees grown in a
heated (≥ 15 °C) greenhouse. During autumn, sap pH was not
directly influenced by temperature. A seasonal change in
H+-ATPase activity resulting from seasonal variation in the
amount of protein was measured in orchard-grown trees,
whereas no significant seasonal changes were recorded in
greenhouse-grown trees. Our data suggest that H+-ATPase
does not regulate xylem sap pH directly by donating protons to
the xylem, but by facilitating secondary active H+/sugar
transport, 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 pH
can vary seasonally, becoming neutral or even alkaline during
some periods of the year. In Acer pseudoplatanus L., xylem
sap pH is close to 6.9 in February (Essiamah 1980) and can
reach 7.5 in Betula pendula Roth by the end of winter (Sauter
and Ambrosius 1986). In species such as Actinidia chinensis L. (Ferguson et al. 1983), Betula pendula (Sauter and
Ambrosuis 1986) and Populus × canadensis “robusta”
(Sauter 1988), pH values are close to neutral in winter and
acidic at the beginning of spring. The acidification mechanism
is not understood, but the cell type best localized to play a major role in the control of xylem sap pH is the vessel-associated
cell (VAC) (Sauter 1988, De Boer and Volkov 2003). Alves et
al. (2001) reported that the structural characteristics of VACs
in walnut are similar to those described for VACs in other species (Läuchli et al. 1974, Van Bel and van der Schoot 1988).
Vessel-associated cells are small, have a high nucleoplasmic
ratio and surround the xylem vessels. They are characterized
by a dense cytoplasm that contains a voluminous nucleus,
small and numerous vacuoles and many organelles and inclusions. Nearly half of the cell surface area in contact with the
vessel has large pits. There are many symplasmic connections
between VACs and rays and axial parenchyma cells (Lachaud
and Maurousset 1996, Alves et al. 2001).
In Robinia pseudoacacia L., seasonal variations in the pH of
solution perfused through the vessels, and the effects on this
pH value of the protonophore carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and of fusicoccin (FC), a specific
activating agent of plasma membrane H+-ATPase (Fromard et
al. 1995), indicate that some living cells in wood tissue are involved in the control of vascular sap pH and that this control
fluctuates seasonally. These patterns are similar to the pH variations occurring in the vascular sap of various woody species
throughout the year and are characterized by a pronounced
acidification in early spring. Essiamah (1980) suggested that
the acidification was a result of the arrival of more acidic sap
from the base of the tree. However, the observed acidification
of tracheal sap in spring could also be explained, at least in
part, by a proton-coupled sucrose efflux into the sap, as reported by Humphreys and Smith (1980) in Zea mays L.
scutellum slices. It has also been suggested that spring acidification of the vascular sap is closely related to plasma membrane H+-ATPase activity (EC 3.6.1.35) of VACs (Fromard et
al. 1995). Moreover, in spring, immunolabeling of plasma
membrane H+-ATPase in VACs has been observed, suggesting
that VACs have enough of this enzyme to control the pH of
vascular sap.
Seasonal changes in plasma membrane H+-ATPase have
been studied during cambial growth in Populus nigra L. and
Populus trichocarpa Torr. & A. Gray (Arend et al. 2002). During autumn and winter dormancy, only a slight immunoreactivity against the plasma membrane H+-ATPase was found
in cross sections and tissue homogenates. In contrast, in spring
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ALVES ET AL.
during cambial growth, strong immunoreactivity was observed in cambial cells and expanding xylem cells. It seems
that this H+-ATPase generates an H+ gradient that can drive the
uptake of K+ and nutrients into cambial and expanding xylem
cells. It was also demonstrated that plasma membrane
H+-ATPase activity varied considerably during the growing
season (Iivonen and Vapaavuori 2002).
The objective of our study was to investigate the possible
role of plasma membrane H+-ATPase of VACs in the control of
xylem sap in walnut trees (Juglans regia L.). The activity of
plasma membrane H+-ATPase was studied by a perfusion
technique, bioluminescence activity assays and Western blot
analyses. The study was performed in winter and early spring
in walnut trees growing in an orchard. Walnut trees kept in a
heated greenhouse (≥ 15 °C) served as a non-hardened reference to characterize the effect of low temperature on the
physiological state of the xylem.
same time every day (0900 h). Around 10 cm of bark from the
apical part of the stem was removed to avoid contamination
with phloem sap. Stems were placed in a vacuum extraction
system allowing simultaneous extraction from several stems
by applying 0.1 MPa suction. Sap samples were collected in
glass tubes placed on ice. Sap pH was measured directly following extraction with a microelectrode (Model N96-3621
Broadley-James, Irvine, CA) During winters 1994 –1995 and
1995–1996, sap pH was determined on two branches of each
of four orchard-grown trees (n = 8 measurements per date). In
winters 1996–1997, 1997–1998 and 1999–2000, measurements were performed on each date on three branches from
each of three trees (n = 9 measurements per date). During winter 1999–2000, sap pH was determined on three branches of
each of two greenhouse-grown trees (n = 6 measurements per
date). Means with standard errors are presented.
Perfusion technique and pH measurements
Materials and methods
Location, climatic data, plant material and treatments
Data were gathered for five winter seasons (1994–1998 and
1999–2000) at the INRA PIAF located near ClermontFerrand, in south-central France. Twenty walnut trees (Juglans
regia cv. Franquette) were grown in orchards and were
13 years old in 1994. Daily maximum and minimum air temperatures were measured at the weather station at Aulnat
(Météo France), which is located less than 1 km away from the
INRA.
Thermal conditioning experiments were performed on container-grown trees. In 1997–1998, 1998–1999 and 1999–
2000, ten 3-year-old walnut trees in individual 33-l pots were
grown outdoors until late summer. The pots contained a 1:2
(v/v) mixture of peat and clay soil that was well-drained and
drip-irrigated to field capacity each day during the growing
season. In late September, the trees were transferred to a
heated greenhouse (temperature between 15 and 25 °C) to ensure that they did not harden during the winter. Because the
greenhouse trees were potted and the outdoor trees were in an
orchard, we irrigated the container-grown trees to field capacity each week to minimize water stress.
Biological measurements
Measurements were performed on excised, 1-year-old twigs
sampled from orchard- and container-grown trees. Each twig
was at least 70 cm long. After removing the uppermost 10-cm
apical part, the sub-adjacent 30-cm-long stem section was
used for the different experiments. It was verified that, when
comparing xylem sap pH values of different samples, between-tree variability was not significantly different from
within-tree variability.
Sap extraction
Xylem sap was extracted from stem segments according to the
method of Bollard (1953) (see Schurr (1998) for a review).
Sap extraction was carried out 1 h after twig excision at the
Twelve-cm-long segments were collected from 1-year-old
twigs after removing the apical 10 cm. Bark was removed
from both ends of each segment and the ends were covered
with paraffin film to prevent dehydration and contamination
with phloem constituents. Three ml of unbuffered standard solution (0.1 mM KCl, 0.1 mM NaCl and 0.1 mM CaCl2 adjusted
to pH 6.0 with 0.01 M HCl or NaOH) was passed through the
stem segments by applying a gentle pressure (0.01 MPa) with
compressed air until a steady-state pH value, called the equilibrium pH, was obtained. The perfusate was unbuffered so
that the VACs would have maximal influence over pH. The effects of the protonophore CCCP (10 µM) and the specific activating agent of the plasma membrane H+-ATPase, FC (10 µM)
were investigated by the perfusion technique described by
Alves et al. (2001). The ∆pH is the difference between the pH
measured in the presence of the effector and the equilibrium
pH.
The pH of the perfusate was measured continuously with a
microelectrode (Type mini combo pH750, World Precision Instruments, Sarasota, FL) inserted into the droplet of the
perfused solution.
Isolation of plasma membrane fraction
The plasma membrane fraction was isolated by a two-phase
aqueous partitioning technique as described by Alves et al.
(2001). All samples were kept on ice throughout the procedure. Briefly, 20 g of xylem (separated from bark, phloem and
cambium) from 1-year-old twigs (three samples of independent stem and independent tree were used for each measurement) was ground in 100 ml of 50 mM HEPES-KOH (pH 7.5)
with 10% (w/v) polyvinylpolypyrrolidine (PVPP), 0.5 M sucrose, 10 mM ascorbic acid, 3.6 mM cysteine, 0.5 mM DTT
and 1 mM phenylmethanesulfonyl fluoride. The slurry was filtered through four layers of cheesecloth and centrifuged for
15 min at 10,000 g. The supernatant was centrifuged for
45 min at 73,000 g. The microsomal fraction was resuspended
in 1 ml of phosphate buffer (5 mM K2HPO4, adjusted to pH 7.8
with KH2PO4) containing 0.3 M sucrose and 3 mM KCl. One
TREE PHYSIOLOGY VOLUME 24, 2004
WINTER VARIATION IN XYLEM SAP pH OF WALNUT TREES
gram of resuspended pellet was added to 14 g of phase mixture
with a final composition of 6% (w/v) Dextran, 6% (w/v) polyethylene glycol (PEG), 1 mM sucrose, 1 mM KCl and 5 mM
phosphate buffer, pH 7.8. After mixing, the system was centrifuged for 5 min at 3000 g. The upper phase was separated, diluted with one volume of Tris buffer (adjusted to pH 6.5 with
MES) containing 0.5 M sucrose and 0.5 mM DTT, and centrifuged for 1 h at 100,000 g. The final pellets, corresponding to
the plasma membrane-enriched fractions, were taken up in
Tris buffer and stored at –80 °C.
Bioluminescence and protein assays
Plasma membrane H+-ATPase activity was measured in 10 µg
of protein by bioluminescence as described by Alves et al.
(2001). We used 0.015% (w/v) Brij 58 detergent to obtain vesicles of uniform sidedness. Plasma membrane H+-ATPase activity was measured at room temperature in 500 µl of 25 mM
Tris-MES buffer, pH 6.5, with 4 mM MgSO4 and 1 mM ATP.
Activity was assayed in the presence of 1 mM NaN3, 50 mM
NaNO3, 0.1 mM Na2MoO4, and in the presence or absence of
500 µM vanadate or 10 µM FC. After 10 min, the ATP remaining was quantified with the Luciferine-luciferase reagent kit
(ATP bioluminescence assay kit CLS II, Roche Diagnostics,
Mannheim, Germany). Plasma membrane proteins were quantified according to Bradford (1976), with bovine serum
albumin as a standard.
101
Results
pH measurements
Xylem sap pH measured during five winter seasons (1994 –
1998 and 1999–2000) showed similar patterns; for simplification, therefore, the mean winter variation for the 5 years is presented in Figure 1A. In orchard-grown walnut trees, xylem sap
pH was close to 7.0 at the beginning of autumn. It dropped rapidly to 5.5 at the beginning of winter and then declined slowly
until the end of February to reach a minimum value of 5.1. In
spring, xylem sap pH increased and a transient alkalinization
was observed in early April in all years investigated. A second
alkalinization period occurred in early May. Moreover, the
seasonal variation in xylem sap pH appeared to parallel the
variation in mean air temperature measured in the orchard. In
trees kept in a greenhouse at 15 °C during winter, xylem sap
pH was close to 6.5 at the beginning of autumn; it decreased
rapidly to 5.4 in early winter, but thereafter no significant
change was observed until April (Figure 1B).
SDS-PAGE and Western blot analyses
A 15-µg sample of plasma membrane proteins was subjected
to SDS-PAGE (polyacrylamide gel electrophoresis) according
to Laemmli (1970). The gel system consisted of a 5% stacking
gel and a 10% resolving gel. Electrophoresis was carried out at
a constant voltage of 150 V for about 3 h.
After gel electrophoresis, polypeptides were transferred
electrophoretically to a nitrocellulose membrane (Transfer
Membrane, BioTraceTM NT, Pall, New York, NY). The transfer buffer contained 25 mM Tris, 193 mM glycin, 0.1% (w/v)
SDS and 20% (v/v) methanol. The transfer was performed at a
constant current of 1 A for 1 h at room temperature.
Tris-buffered saline was used as the basic medium for
immunoblotting. Teleostan gelatin (Sigma, St. Louis, MO) 3%
(w/v) and Tween 20 (0.75 ml l –1) were used in the blocking of
nitrocellulose filters. A rabbit immunopurified antiserum
raised against a peptide of the central domain of the plasma
membrane H+-ATPase (peptide CDPKEARAGIREVHF) was
diluted 1/2000, and the second antibody, conjugated to alkaline phosphatase, was diluted 1/3000 (monoclonal anti-rabbit
IgG alkaline phosphatase conjugate, immunoglobulin fraction
of mouse ascites fluid, clone RG96 from Sigma). The alkaline
phosphatase substrates were nitroblue tetrazolium (NBT) and
5-bromo-4-chloro-5-indolyl phosphatase (BCIP). The bands
on the western were quantified by image analysis (Bio-1D
software, Vilber Lourmat, Marne-la-vallée, France).
Figure 1. (A) Autumn, winter and spring variations in air temperature
and vascular sap pH of orchard-grown walnut trees. Data shown are
means ± SE for 5 years from October 1994 to May 2000. Sap pH was
measured twice monthly on 1-year-old twigs. (B) Autumn, winter and
spring variations in vascular sap pH of walnut trees grown in an orchard 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 of
the plasma membrane H+-ATPase of VACs on xylem sap pH.
After perfusing 3 ml of standard solution, the pH of the
perfused solution stabilized to a constant value (equilibrium
pH), indicating that the stem segment was totally rinsed of its
original sap. The equilibrium pH (mean value of the 3 years
1998–2000) is presented in Figure 2A. In orchard-grown
trees, the equilibrium pH of the solution perfused was 5.3 in
February and close to 5.7 in April. For the same periods, no
significant change of the equilibrium pH value (5.7) was observed in trees kept in a greenhouse at 15 °C during winter.
After the pH stabilized to its equilibrium value, the perfusing 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 alkalinization
of the perfusing liquid in April (0.28 pH unit) than in February
(0.18 pH unit), revealing seasonal variations in the pH gradient
between the cytoplasm of VACs and xylem sap. A similar pattern was observed in container-grown trees in the greenhouse.
In orchard-grown trees, the FC treatment performed in Febru-
Figure 2. Winter and spring variations in pH of the solution perfused
through 1-year-old stem segments harvested from walnut trees grown
in an orchard or kept in a greenhouse at ≥ 15 °C. Experiments were
performed in 1998, 1999 and 2000. (A) Seasonal variations in the
equilibrium pH. Mean equilibrium pH values ± SE (n ≥ 30) are reported. (B) Effects of cyanide-m-chloro-phenylhydrazone (CCCP)
(white bar) and fusicoccin (FC) (solid bar) on the pH of the perfused
solution, after equilibrium. Mean ∆pH values ± SE (n ≥ 15) are reported.
ary had no significant effect on the pH of the solution perfused,
whereas FC induced acidification of about 0.33 pH units in
April, suggesting that VACs have enough plasma membrane
H+-ATPase to control xylem sap pH. In greenhouse-grown
trees, FC induced significant acidification of about 0.19 and
0.10 in February and April, respectively.
Plasma membrane H+-ATPase activity and quantification
The purity of the plasma membrane-enriched fraction was estimated with various ATPase effectors (data not shown). In xylem 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 fractions
were free from mitochondria and thylakoid contamination.
In xylem tissue of orchard-grown trees, the specific activity
of H+-ATPase increased from 6 to 30 nKat (mg protein) –1
from February to April (Figure 3A). Addition of FC increased
the H+-ATPase activity by 9% relative to the control in February and by 46% in April (Figure 3B). In trees kept in the greenhouse, H+-ATPase specific activity increased from 29 to
35 nKat (mg protein) –1 from February to April (Figure 3A).
Addition of FC increased H+-ATPase activity by 23% relative
Figure 3. (A) Plasma membrane H+-ATPase specific activity measured 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 of
xylem plasma membrane H+-ATPase activity by fusicoccin (FC) in
1999 and 2000. Each value is the mean of five measurements ± SE.
TREE PHYSIOLOGY VOLUME 24, 2004
WINTER VARIATION IN XYLEM SAP pH OF WALNUT TREES
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 walnut
stem in the 100 kDa region (Figure 4). In February, the amount
of plasma membrane H+-ATPase of xylem was higher in the
trees kept in the greenhouse than in the orchard-grown trees
(Figure 4). In the orchard-grown trees, the amount of plasma
membrane H+-ATPase was higher in April than in February.
The amount of plasma membrane H+-ATPase in the trees kept
in the greenhouse was high and did not change between February and April. Moreover, it seemed that, in April, the amount
of plasma membrane H+-ATPase was similar in orchard- and
greenhouse-grown trees.
Discussion
Seasonal variation in xylem sap pH and the possible involvement of the plasma membrane H+-ATPase in the control of
vascular sap pH were investigated in walnut trees. In orchard-grown trees (Figure 1A), xylem sap pH was close to
neutral in October, then decreased rapidly to acidic values until February, and a transient alkalinization was systematically
Figure 4. Changes in amount of xylem plasma membrane H+-ATPase
in walnut trees grown in an orchard or kept in a greenhouse in winter
and spring of the 1999–2000 season. Upper panel: Western blot of the
plasma membrane H+-ATPase (Mr ≈ 100 kDa) from 1-year-old stems
of walnut xylem tissue. Lanes 1 to 4 were loaded with 15 µg protein.
Molecular mass markers (kDa) are indicated. Lower panel: Each
value represents the mean band intensity (± SE) of three replicates.
103
observed at the beginning of April. These results suggest that
the seasonal variation in xylem sap pH is correlated to temperature as reported by Fromard (1990) in Acer platanoides L. for
temperatures below zero. However, we observed (Figures 1A
and 1B) that xylem sap pH decreased rapidly to acidic values
during autumn in both orchard- and greenhouse-grown trees,
indicating that, at least during this period, sap pH was not directly influenced by temperature.
In contrast to walnut trees, sap pH in many woody species is
close to neutral in winter and becomes acidic (pH 5.5) at the
beginning of spring (Ferguson et al. 1983, Sauter and
Ambrosius 1986, Sauter 1988, Fromard 1995). We used several methods to investigate the role of H+-ATPase in the regulation of vascular sap pH in walnut trees. The use of a
perfusion technique allowed continuous monitoring of the
variation in pH of the perfused solution. In orchard-grown
trees, the pH gradient revealed by use of CCCP and the amplitude of the acidification caused by FC showed seasonal variations, with the responses being highest in April and lowest in
February (Figure 2B). This pattern in H+-ATPase activity
could be mainly explained by seasonal variation in the amount
of protein. A similar seasonal variation in the amount of the
plasma membrane H+-ATPase was recently reported during
cambial growth in Populus nigra and Populus trichocarpa
(Arend et al. 2002). Although it is clear that VACs represent a
minor component of the living cells of xylem in branches compared with other parenchyma cells, it has been demonstrated
that immunostaining of the plasma membrane H+-ATPase is
much stronger in VACs than in other living cell types of the
xylem (Fromard et al. 1995).
The equilibrium pH values (Figure 2A) of the perfused solution showed similar variations to those measured directly in
sap collected by vacuum extraction (Figure 1). The difference
between the pH of crude sap and the equilibrium pH of the
perfused solution revealed short-term pH modifications of the
standard solution. The acidification of the perfused solution in
February in the orchard-grown trees (Figure 2A) confirmed
the capacity of the living cells of the xylem tissue to modify
vascular sap pH. In Robinia pseudoacacia, Fromard et al.
(1995) proposed that the plasma membrane H+-ATPase of
VACs could control vascular sap pH and suggested that spring
acidification of the vascular sap was closely related to the activity of the enzyme. However, our observation that winter
acidification occurred in the perfusate (Figure 2A) of the orchard-grown trees when H+-ATPase activity was lowest (Figure 3A) casts some doubt on the validity of this hypothesis for
walnut. Our observation is supported by the study of Gerendas
and Schurr (1999) who observed that proton concentration itself has little influence on xylem sap pH. Furthermore, we
have obtained circumstantial evidence for the existence of an
alternative non-H+-ATPase mechanism for the acidification of
xylem sap in walnut in winter.
As recommended by Gerendas and Schurr (1999), a detailed
analysis of the composition of the xylem sap is necessary to
identify which compound(s) among organic ions, inorganic
ions, amino acids and partial pressure in CO2 might be responsible for the winter acidification of xylem sap. As late as No-
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ALVES ET AL.
vember, positive pressures were detected in intact plants but
not in excised segments (Ewers et al. 2001). These authors
concluded that autumn pressures appeared to be of root origin
and demonstrated that they were associated with the uptake of
mineral nutrients from soil, especially nitrate. In February, the
stem pressure appeared to be of stem origin, resulting in the
accumulation of sugars in the sap.
In walnut, xylem sugar accumulation is correlated with the
repair of winter xylem embolism, resulting in xylem vessel
conductivity restoration (Améglio et al. 1995, Améglio et al.
2002). Xylem sugar accumulation results from the balance of
two opposite movements: an efflux of sugars from parenchyma cells into the xylem vessels (Sauter 1980) and an influx
of sugars from the xylem sap into VACs (Sauter 1981). Current
hypotheses about the mechanism for both movements include
facilitated diffusion for the efflux (Sauter 1982) and an
H+/sugar symport for the influx (Fromard 1990). This influx
could be dependent on plasma membrane H+-ATPase. In walnut, the efflux of sugar seems to drive local water movements
(Lacointe et al. 1995). However, the mechanisms of the two
fluxes remain hypothetical. It is possible that the sugar efflux
occurring in winter in walnut is responsible for sap acidification.
Moreover, the nonsignificant acidification of the perfusate,
measured in April in the orchard-grown trees, and the acidification in both winter and spring in trees kept in the greenhouse, when H+-ATPase activity was high, confirms that the
vascular sap pH does not depend directly on an increase in protons being pumped into the xylem vessels.
In early spring, sap pH of the orchard-grown trees increased
transiently (Figure 1A) when H+-ATPase activity was high
(Figure 3A). Because our trees were well-watered, it seems
unlikely that this increase in sap pH occurred in response to a
drought signal as previously reported in Commelina communis L. by Wilkinson and Davies (1997). We propose that
this alkalinization is associated with secondary active H+-coupled cotransports. It is assumed that phloem is inactive during
winter and that sugar mobilization at bud break occurs through
the xylem pathway (Sauter and Ambrosius 1986, Lacointe et
al. 2001). The increase in H+-ATPase activity at the beginning
of spring could permit massive sugar uptake via an H+/sugar
symport in the VACs (Fromard 1990) leading to the alkalinization of the sap pH.
In the walnut trees kept in the greenhouse, no seasonal variations in equilibrium pH were observed (Figure 2A). Furthermore, neither the H+-ATPase activity (Figure 3A) nor the
amount of protein (Figure 4) showed a significant seasonal
variation. Compared with the orchard-grown trees, these data
indicated that winter temperatures had an effect on H+-ATPase
activity. In the trees kept in the greenhouse, the high ATPase
activity measured throughout the winter (Figure 3) would be
energy-consuming, leading to the depletion of reserves that
could account for the erratic bud break reported by Améglio
and Cruiziat (1992). These results also indicate the need to
study reserve mobilization in cold-deprived trees.
To summarize, we showed seasonal variation in xylem sap
pH of walnut trees and demonstrated that this pattern is not di-
rectly attributable to protons contributed by ATPase. Further
studies are necessary to elucidate the origin of the winter acidification of xylem sap, the efflux mechanism and to confirm the
involvement of a secondary active H+/sugar influx in spring
alkalinization.
Acknowledgments
We thank C. Bodet, S. Ploquin and P. Chaleil for their technical assistance. The skillful help of M. Bonhomme, N. Brunel and C. Beaudoin
is gratefully acknowledged.
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