Download Seasonal Changes of Plasma Membrane H

Seasonal Changes of Plasma Membrane Hⴙ-ATPase and
Endogenous Ion Current during Cambial Growth in
Poplar Plants1
Matthias Arend*, Manfred H. Weisenseel, Maria Brummer, Wolfgang Osswald, and Jörg H. Fromm
Fachgebiet Angewandte Holzbiologie (M.A., J.H.F.) and Fachgebiet Pathologie der Waldbäume (M.B., W.O.),
Technische Universität München, Munich, Germany; and Botanisches Institut, Universität Karlsruhe,
Karlsruhe, Germany (M.H.W.)
The plasma membrane H⫹-ATPase (PM H⫹-ATPase), potassium ions, and endogenous ion currents might play a fundamental role in the physiology of cambial growth. Seasonal changes of these parameters were studied in twigs of Populus nigra
and Populus trichocarpa. Monoclonal and polyclonal antibodies against the PM H⫹-ATPase, x-ray analysis for K⫹ localization
and a vibrating electrode for measurement of endogenous ion currents were used as probes. In dormant plants during
autumn and winter, only a slight immunoreactivity against the PM H⫹-ATPase was found in cross sections and tissue
homogenates, K⫹ was distributed evenly, and the density of endogenous current was low. In spring during cambial growth,
strong immunoreactivity against a PM H⫹-ATPase was observed in cambial cells and expanding xylem cells using the
monoclonal antibody 46 E5 B11 F6 for fluorescence microscopy and transmission electron microscopy. At the same time, K⫹
accumulated in cells of the cambial region, and strong endogenous current was measured in the cambial and immature
xylem zone. Addition of auxin to dormant twigs induced the formation of this PM H⫹-ATPase in the dormant cambial region
within a few days and an increase in density of endogenous current in shoot cuttings within a few hours. The increase in
PM H⫹-ATPase abundance and in current density by auxin indicates that auxin mediates a rise in number and activity of
an H⫹-ATPase in the plasma membrane of cambial cells and their derivatives. This PM H⫹-ATPase generates the necessary
H⫹-gradient (proton-motive force) for the uptake of K⫹ and nutrients into cambial and expanding xylem cells.
The plasma membrane H⫹-ATPase (PM H⫹ATPase) is a key enzyme of plants and fungi, and it
plays a central role in nutrient uptake and growth of
higher plants (Serrano, 1989; Palmgren, 2001). The
enzyme generates the proton-motive force that drives
the uptake of nutrients such as sugars and ions across
the plasma membrane of growing plant cells. Especially the uptake of potassium ions through specific
transport proteins has been related to the activity of
the PM H⫹-ATPase (Hoth et al., 1997; Maathuis et al.,
1997). This uptake is essential for osmotic regulation
and cell enlargement in differentiating tissues (MacRobbie, 1977; Hsiao and Läuchli, 1986). Most of the
information concerning the PM H⫹-ATPase has been
obtained from biochemical and histochemical studies
on herbaceous plant species, where the enzyme was
found predominantly in vascular tissue, in companion cells of the phloem, in epidermal cells of roots,
and in guard cells of leaves (compare with Palmgren,
1998). The results revealed an important role of the
enzyme in phloem loading, uptake of minerals by
roots, and turgor regulation in guard cells.
1
This work was supported by the Deutsche Forschungsgemeinschaft (grant no. FR 955/3–1,2).
* Corresponding author; e-mail [email protected];
fax 49 – 89 –2180 – 6429.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.003905.
In contrast to herbaceous plants, very little is
known about the occurrence of this vital enzyme in
woody plants or about its distribution and function
in individual tissues. Some work has been done regarding enzyme activities of vessel-associated cells in
walnut trees (Alves et al., 2001) or the variation of
different PM H⫹-ATPase transcripts in buds of peach
(Prunus persica) trees during breaking of dormancy
(Gevaudant et al., 2001). Because of the strong demand for minerals by woody plants during the time
of cambial growth, an involvement of the PM H⫹ATPase in the process of wood formation is expected.
The cambial region acts as a strong axial sink that
competes for minerals and assimilates with other
sinks such as young leaves and roots during the time
of wood formation (Dünisch and Bauch, 1994; Krabel,
2000). The energy for the uptake of the solutes into
growing cambial cells is most likely provided by the
proton-motive force generated by the PM H⫹ATPase (Michelet and Boutry, 1995). Especially the
supply and uptake of potassium ions might affect the
process of wood formation due to the significance of
turgor-driven cell enlargement of cambial cell
derivatives.
The acid growth hypothesis holds that susceptible
cells exposed to auxin secrete protons into the apoplast, which causes hyperpolarization of the cells, a
decrease in apoplastic pH, and an increase in the
growth rate (Rayle and Cleland, 1992). This hyper-
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Arend et al.
polarization and the apoplastic acidification drive the
uptake of nutrients into cells via secondary active
transport and cause the opening of ion channels, for
instance K⫹ channels (Michelet and Boutry, 1995;
Hoth et al., 1997; Maathuis et al., 1997). With respect
to trees, numerous studies have shown that auxin is
an important regulator of cambial growth (Savidge
and Wareing, 1981; Little and Savidge, 1987; Lachaud,
1989; Little and Pharis, 1995; Uggla et al., 1998; Sundberg et al., 2000). However, our understanding of the
cellular events is still poor, and no effect of auxin on
the PM H⫹-ATPase in cambial cells has been demonstrated so far.
In poplar (Populus spp.) plants, with their fastgrowing trunks, shoots, and twigs, a PM H⫹-ATPase
may have a vital role in the uptake of nutrients by
cambial and other growing cells. Suc has been shown
to be the major carbohydrate of cambial metabolism
(Krabel, 2000). During the period of rapid cell divisions and cell growth in spring and summer, the
demand for Suc reaches its peak, and the number and
activity of a PM H⫹-ATPase might be high to energize the uptake of Suc into cells. Some support for an
involvement of a PM H⫹-ATPase during cambial
growth comes from investigations showing differences in electric membrane potential among the various cell types involved in assimilate flux from sieve
tubes to cambial cells (van Bel and Ehlers, 2000).
Moreover, transport of assimilates to the cambium
has been demonstrated directly in willow (Salix viminalis), i.e. close relatives to poplar plants, using labeled sugars and microautoradiography (Fromm,
1997). Circumstantial evidence indicating the cambial
cells as the major sink for nutrients is derived from
the large number of plasmodesmata in tangential
walls of ray cells (Sauter and Kloth, 1986).
In this study, various methods were applied to
investigate seasonal changes and an assumed effect
of auxin on the PM H⫹-ATPase of cambial cells: (a)
Changes in potassium distribution were measured by
x-ray microanalysis. (b) Specific antibodies against
the plasma membrane H⫹-ATPase were used to immunolocalize the enzyme to measure its distribution
and amount in thin sections of Populus trichocarpa
twigs. Either the antibody was labeled with Cy3 and
localized by fluorescence microscopy, or it was labeled with gold particles and visualized by transmission electron microscopy. (c) Tissue homogenates
were analyzed using the western-blot technique for
the presence of PM H⫹-ATPase at various times of
the year and in dormant plants after the addition of
auxin. (d) Endogenous ionic current as an indicator
of the activity of a PM H⫹-ATPase was measured
with a noninvasive, vibrating electrode near the face
of stem cuttings from shoots of Populus nigra, assuming that the PM H⫹-ATPase drives H⫹ current
through the plasma membrane and the adjacent apoplast. Auxin was either applied briefly to measure
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short-term effects of the hormone or for several days
to allow for gene activation.
RESULTS
Potassium Distribution
Energy-dispersive x-ray analysis in combination
with scanning electron microscopy (SEM) provides
an appropriate technique for semiquantitative detecting of elements in the intact tissue on the microscopic
level. Although the physical heterogeneity of the tissue matrix did not permit a comparison with a calibration standard for absolute quantification of potassium concentrations, this method revealed a strong
seasonal change of the relative potassium concentration and distribution in twig tissue of P. trichocarpa.
Especially the cambial region showed strong changes
in potassium content reflecting its seasonal states of
activity: An increased level of potassium was measured in the active cambial region in May, whereas
only a low level of potassium was found in the
dormant cambium and its daughter cells in January
(Fig. 1). In comparison with the adjacent mature tissues of the phloem and xylem, the highest amount of
potassium was found in the active cambial region as
indicated by the distinct accumulation of potassiumspecific x-ray signals in this part of the twigs (Fig. 2,
C and D). In contrast, potassium showed an equal
distribution across twigs during the time of cambial
dormancy (Fig. 2, A and B).
Western-Blot Analysis
The specificity of the monoclonal antibody 46 E5
B11 F6 against a PM H⫹-ATPase of P. trichocarpa was
checked by western-blot analysis of twig tissue homogenates. After electrophoresis of twig tissue homogenate collected in May after cambial reactivation,
a binding of the antibody to a polypeptide in the
Figure 1. Relative potassium concentrations in dormant (January)
and active (May) cambium cells from P. trichocarpa expressed as the
peak-to-background ratio of element-specific x-ray signals. Values
are mean ⫾ SE from measurements of 10 different tissue areas.
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Plant Physiol. Vol. 129, 2002
Plasma Membrane H⫹-ATPase in Poplar
Figure 2. Potassium distribution in twigs of P. trichocarpa. A, SEM micrograph of a transverse section taken during cambial
dormancy (November). B, Mapping of potassium-specific x-ray signals in the same twig section as shown in A. C, SEM
micrograph of a transverse twig section taken after cambial reactivation (May). D, Mapping of potassium-specific x-ray
signals in the same twig section as shown in C, indicating a strong accumulation of potassium in the activated cambial zone
(arrows).
100-kD range was detected on the blots (Fig. 3A). A
second minor band was recognized by the antibody
in the 60-kD range, probably a proteolytic degradation product of the enzyme with an epitope for the
antibody. In contrast, no PM H⫹-ATPase was detected by the antibody on blots that were prepared
from twig tissue in November during cambial dormancy (Fig. 3A). Auxin applied for 2 d to debudded
twigs (40 ␮m indole-3-acetic acid [IAA]-sodium salt
in water) during the period of dormancy induced a
new expression of PM H⫹-ATPase as indicated by
immunodetectable amounts of the enzyme on the
blots (Fig. 3B). A polyclonal antibody raised against a
PM H⫹-ATPase from Nicotiana plumbaginifolia reacted with twig tissue homogenate from poplar
plants similarly to the monoclonal antibody 46 E5
B11 F6 (Fig. 3C). During cambial activity a major
band was detected by the polyclonal antibody in the
100-kD range and a second minor band in the 60-kD
range, whereas twig tissue prepared during cambial
dormancy showed only a very slight immunoreactivity in the 100-kD range.
Plant Physiol. Vol. 129, 2002
Immunofluorescence Microscopy
Cross sections were cut from twigs in spring after
cambial reactivation and incubated with the monoclonal antibody 46 E5 B11 F6 against PM H⫹-ATPase
and a Cy3-conjugated secondary antibody. A strong
specific fluorescence labeling was visible predominantly in the active cambial zone and the adjacent
region of xylem cell differentiation (Fig. 4A). In the
mature xylem, there was some specific labeling of ray
cells that contacted vessel cells (Fig. 4B). In contrast,
no specific labeling was observed in cross sections
taken during cambial dormancy (Fig. 4C). After treatment of debudded twigs in December during cambial
dormancy with 40 ␮m IAA for 4 d, specific labeling of
a PM H⫹-ATPase became visible in the cambial zone
and in some ray cells that contact vessels in the
mature xylem (Fig. 4, D and E). Controls showed
only an unspecific background labeling, either in the
absence of the primary antibody or after replacement
of the specific antibody with nonimmune IgG (Fig.
4F). For comparison, twig sections were also treated
with the polyclonal antibody L3E against PM H⫹-
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1653
Arend et al.
Figure 3. Western blots of twig tissue homogenates from P. trichocarpa. A, Blot incubated
with the monoclonal antibody 46 E5 B11 F6.
Samples taken from twig tissue during cambial
activity (May), respectively, cambial dormancy
(November). B, Blot incubated with the monoclonal antibody 46 E5 B11 F6. Samples taken
during cambial dormancy (November) from
twig tissue treated with auxin for 2 d, respectively, without auxin. C, Blot incubated with the
polyclonal antibody L3E. Samples taken from
twig tissue during cambial activity, respectively,
cambial dormancy. Molecular mass markers are
indicated on the left.
ATPase in place of the monoclonal antibody. Sections
taken during cambial activity showed a fluorescence
pattern different from that given with the monoclonal antibody. A distinct specific labeling occurred in
cells of the inner phloem, the transition zone phloem/cambium, and the ray parenchyma, whereas no
PM H⫹-ATPase was detected by the polyclonal antibody in the differentiating xylem (Fig. 5A). In addition, a weak specific fluorescence was visible in many
cells of the cortex, indicating the presence of a small
amount of PM H⫹-ATPase in this tissue (Fig. 5B).
Twig sections taken during cambial dormancy
showed a reduced intensity of the fluorescence labeling, mostly restricted to ray cells crossing the cambial
zone and the inner phloem (Fig. 5C). Controls
showed no fluorescence, either in the absence of the
primary antibody or after replacement of the specific
antibody with nonimmune IgG (Fig. 5D).
Immunogold Electron Microscopy
Because the specific fluorescence labeling given by
the monoclonal antibody appeared all over the labeled cells, immunohistochemical experiments were
carried out with the transmission electron microscope for detailed localization of this PM H⫹-ATPase.
A procedure of labeling before embedding was carried out using ultrasmall gold conjugates in combination with silver enhancing. The silver-enhanced
gold particles appeared predominantly along the
plasma membrane of labeled cells, indicating that a
large number of the PM H⫹-ATPases was present in
this cellular domain (Fig. 6, A–F). In agreement with
results obtained from immunofluorescence microscopy, a specific labeling of a PM H⫹-ATPase was
found along the plasma membrane of cells of the
active cambium, as well as in expanding cells of the
immature xylem and in ray cells contacting vessels of
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the mature xylem. In cambial cells, the intensity of
immunolabeling along the plasma membrane was
always higher in radial sections than in cross sections
because of the much better preservation of cellular
structures, including membranes, in the former case
(Fig. 6, A and B). Differentiating xylem cells, newly
formed vessels, fiber cells, and ray cells showed labeling of a lower intensity than the cambial cells
proper (Fig. 6, C–F). A very strong immunolabeling
of the plasma membrane was observed in companion
cells of the inner phloem in contact with the active
cambium (Fig. 7C), whereas the sieve elements attached to these companion cells showed no labeling.
The transmission electron microscopy results also
show that a labeling of ray cells in mature xylem only
occurs when these cells contact vessels (Fig. 7, A and
B). A few gold particles were bound to other cell structures. The latter labeling was nonspecific, because a
similar pattern was observed in control sections incubated with nonimmune IgG (Fig. 7, D and E).
Endogenous Ionic Current during the Seasons
The direction and density of endogenous ionic current was measured at different sites of cross sections
during various times of the year. The results show
that the current pattern and current density in shoot
cuttings of P. nigra depended on the season: In dormant shoots in December, only small outward current was measured at all sites, i.e. the pith parenchyma, secondary xylem (wood), cambium/phloem
zone, and cortex parenchyma (Fig. 8). In April, a
period of active cambial growth, strong outward current with densities of more than 6 ␮A cm⫺2 was
measured in the cambium/phloem zone and the
green cortex. Outward current was also found in the
pith and the mature xylem, whereas strong inward
current with densities of up to 8 ␮A cm⫺2 was mea-
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Plant Physiol. Vol. 129, 2002
Plasma Membrane H⫹-ATPase in Poplar
Figure 4. Localization of a PM H⫹-ATPase in twig tissue of P. trichocarpa using the monoclonal antibody 46 E5 B11 F6. A,
Section taken after cambial reactivation (April). Strong fluorescence labeling appears in the cambium zone (arrows) and the
adjacent region of xylem cell differentiation. Arrowheads show newly formed vessels. B, Section taken after cambial
reactivation. Fluorescence labeling of some ray cells that contact vessels in the mature xylem (arrows) can be seen. C,
Section taken during cambial dormancy (December). No fluorescence labeling of a PM H⫹-ATPase is visible. D, Section
taken during cambial dormancy (December) after treatment of debudded twigs with auxin for 4 d. Fluorescence labeling
appears in the cambium/phloem zone (arrows). E, Fluorescence labeling of ray cells in the mature xylem after treatment of
debudded twigs with auxin in December (arrow). F, Control section taken after cambial reactivation (April) incubated with
nonimmune IgG instead of the monoclonal antibody against a PM H⫹-ATPase. Xy, Xylem; Ph, phloem.
sured in the young xylem. A similar current pattern
was measured in August, but the current direction in
the cambium/phloem zone had changed to inward.
The overall current density decreased from April to
August and from August to December at all measurement sites.
Plant Physiol. Vol. 129, 2002
During experiments performed in August, the
ionic composition of the medium was changed to
obtain information about the ions involved in the
endogenous current. When the pH of the medium
was increased, the current density also increased significantly at all measurement sites, and outward cur-
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1655
Arend et al.
Figure 5. Localization of the PM H⫹-ATPase in twig tissue of P. trichocarpa using the polyclonal antibody L3E. A, Section
taken after cambial reactivation. Fluorescence labeling appears in the inner phloem and the transition zone phloem/
cambium (arrows). No specific labeling occurs in the differentiating xylem (arrowheads). B, Slight fluorescence labeling of
cortex cells in a section taken after cambial reactivation (arrows). C, Section taken during cambial dormancy, with slight
fluorescence labeling of cells in the inner phloem. Arrows indicate ray cells crossing the cambium and the inner phloem.
D, Control section taken after cambial reactivation and incubated with nonimmune IgG instead of the polyclonal antibody
against a PM H⫹-ATPase. Xy, Xylem; Ph, phloem; Scl, sclerenchyma.
rent became dominant. Figure 9 shows a typical example of current density vectors measured across the
face of cuttings, first at pH 4.5 and then at pH 7.5. At
both sites, i.e. the cortex parenchyma (Fig. 9A) and
the cambium/phloem zone (Fig. 9B), the current density increased, and the direction changed from inward to outward in the cambium/phloem zone. No
immediate changes of the current density were measured when the potassium or chloride concentration
of the medium was changed (data not shown). After
increasing the calcium concentration of the medium
to 1.0 mm, a decrease of the outward current was
observed at the cortex and a decrease of the inward
current in the cambium/phloem zone.
Because it is well known that plant hormones are
involved in cambial growth and differentiation of
trees, we investigated the effects of two prominent
phytohormones, i.e. IAA and abscisic acid (ABA), on
the endogenous ionic current of poplar shoots. The
effect of 30 ␮m IAA was measured in December
when only small endogenous current was present.
In accord with the IAA activation of a PM H⫹1656
ATPase shown with immunoblot and immunofluorescence (compare with Figs. 3 and 4), IAA caused
an increase of current density at all measurement
sites already 2 h after application (Fig. 10A). The
mean current density increased by 60% from 1.7 to
2.8 ␮A cm⫺2.
The formation of latewood at the end of summer
has been attributed to an increase in the level of ABA
in the cambial zone (Jenkins and Shephard, 1974;
Wodzicki and Wodzicki, 1980). ABA was also shown
to have a strong inhibitory effect on wood growth
(Fromm, 1997) and magnitude of endogenous current
of willow roots (Fromm et al., 1997). During the time
of cambial activity and high-current densities in
spring, application of 30 ␮m ABA to the medium
caused the density of outward current to decrease by
almost 50% within 2 h, i.e. from 6.2 to 3.2 ␮A cm⫺2
(Fig. 10B). These results suggest an opposing role of
both hormones in the regulation of endogenous current and activity of the PM H⫹-ATPase activity in
poplar plants.
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Plant Physiol. Vol. 129, 2002
Plasma Membrane H⫹-ATPase in Poplar
Figure 6. Cellular fine localization of a PM H⫹-ATPase in the active cambium zone and the region of xylem cell
differentiation using the monoclonal antibody 46 E5 B11 F6. Samples were taken in May. A and B, Radial section of an active
cambial cell with strong labeling along the plasma membrane (arrows). C and D, Cross section of newly formed xylem cells.
Labeling of the plasma membrane appears in a ray cell and a fiber cell (arrows). E and F, Radial section of an enlarging vessel
cell with labeling of the plasma membrane (arrows). V, Vacuole; CW, cell wall; VC, vessel; RC, ray cell; FC, fiber cell.
Plant Physiol. Vol. 129, 2002
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Arend et al.
Figure 7. Cellular fine localization of a PM H⫹-ATPase in the inner
phloem and in ray cells of mature xylem. Samples were taken in May.
A and B, Cross section of mature xylem. Labeling appears along the
plasma membrane of a ray cell that contacts a mature vessel cell
(arrows). C, Cross section of a sieve element/companion cell complex of the inner phloem. Strong labeling occurs along the plasma
membrane of the companion cell. D and E, Controls. Cross sections
of a ray cell and a cambial cell. VC, Vessel; RC, ray cell; CA, cambial
cell; SE, sieve element; CC, companion cell.
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Plant Physiol. Vol. 129, 2002
Plasma Membrane H⫹-ATPase in Poplar
Figure 8. Pattern and densities (⫾SE) of endogenous ionic current of
different tissues across sections of vertically oriented shoot cuttings
from P. nigra measured in April, August, and December. Current
densities were measured in more than 10 different shoots collected
from the trunks or roots of P. nigra trees.
DISCUSSION
In this study, the PM H⫹-ATPase, K⫹-ion content
and endogenous ionic currents were investigated in
poplar plants using specific antibodies, x-ray analysis, and a noninvasive vibrating probe. A PM H⫹ATPase was visualized by the monoclonal antibody
46 E5 B11 F6 in a very sensitive manner in physiologically active poplar plants during cambial growth,
but not in dormant ones. In contrast to herbaceous
species, this PM H⫹-ATPase was localized in the
cambium zone, in differentiating xylem cells and in
ray cells surrounding vessels in the mature xylem.
Auxin applied to dormant plants induced the formation of this PM H⫹-ATPase in the cambial region as
well as in ray cells. Experiments carried out additionally with the electron microscope showed that the
PM H⫹-ATPase was localized in the plasma membrane of the labeled cells. No immunoreactivity was
obtained with this antibody in the cortex parenchyma and in pith cells. This result came as a surprise
because these tissues produced ionic currents with
similar direction and density as the cambial region
during periods of high-growth activity. Using a polyclonal antibody, further PM H⫹-ATPases were localized in the inner phloem and in cells of the cortex
parenchyma. Surprisingly, no immunoreactivity was
found using the polyclonal antibody in the differentiating xylem and in pith cells. Only a slight labeling
was observed during cambial dormancy.
Taken together, a clear seasonal variation in the
amount of the PM H⫹-ATPase was shown in poplar
Plant Physiol. Vol. 129, 2002
twigs using different specific antibodies and correlated to cambial growth activity. Similar to this observation, a seasonal variation of different PM H⫹ATPase transcripts was demonstrated recently in the
bud tissue of peach trees (Gevaudant et al., 2001). The
different labeling pattern given by monoclonal and
polyclonal antibodies indicated that different isoforms of the enzyme are present in poplar tissues, of
which one group is mainly found in the cambial
tissue and the differentiating xylem. Molecular studies have shown that in plants, the PM H⫹-ATPase is
encoded by a multigene family, and PM H⫹-ATPase
isoforms exhibit a distinct pattern of tissue-specific
expression (compare with Palmgren, 1998; Palmgren,
2001). For castor bean (Ricinus communis) plants, it
was recently demonstrated that the monoclonal antibody 46 E5 B11 F6 discriminates between different
PM H⫹-ATPase isoforms in a cell-specific manner
(Langhans et al., 2001). The complete lack of specific
labeling for a PM H⫹-ATPase in pith cells remains an
open question, because this tissue drove ionic currents during periods of high-growth activity. Besides
the occurrence of further PM H⫹-ATPase isoforms,
an alternative explanation for the outward current at
the pith might be an efflux of K⫹ and Na⫹ or an
influx of Cl⫺ ions. K⫹ and Na⫹, however, are very
unlikely to carry the outward current. A massive and
prolonged leakage of K⫹ from the small number of
injured cells at the cutting plane does not seem feasible, and a Na⫹/K⫹ ATPase that would transport
Na⫹ to the medium is absent in plant cells. Net Cl⫺
uptake, on the other hand, might be a real possibility
for the outward current. Such uptake has been reported from bean leaves during photosynthesis (Shabala and Newman, 1999), and pith cells showed a
distinct fluorescence of chlorophyll when twig sections from poplar plants were inspected with the
fluorescence microscope.
During all seasons, the cortex parenchyma, the mature xylem, and the pith parenchyma drove current
from the protoplasts to the apoplast. The cambial
zone showed outward current in the spring, but inward current in the late summer. Young wood cells
were a current sink during the entire growing season.
The current densities were highest in spring, declined in late summer, and became rather small in
winter when the plants were dormant.
Combining the data from PM H⫹-ATPase localization and current measurements, there are several
indicators that a major component of the endogenous
current is H⫹ ions: (a) The high concentration of PM
H⫹-ATPases, particular in the cambial zone and the
differentiating xylem, during cambial growth correlates positively with outward current during the
same time. The slight amount of immunodetectable
PM H⫹-ATPases in dormant plants conversely correlates with low-current densities. (b) The current density increased with increasing pH and addition of
auxin. A higher pH on the outside of cells favors H⫹
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Arend et al.
Figure 9. Typical examples of the changes in
current density and direction after increasing the
pH of the medium from pH 4.5 to 7.5. A, Endogenous current at the cortex parenchyma of a
poplar shoot cutting. B, Endogenous current at
the cambium/phloem zone. Both examples were
measured in August in shoots from P. nigra trees.
export by the PM H⫹-ATPase (Takeuchi et al., 1985;
Takeshige et al., 1986), and auxin is known to stimulate H⫹ excretion (Talbott et al., 1988; Lüthen et al.,
1990; Hager et al., 1991). (c) The current density
decreased with increasing Ca2⫹ concentration and
addition of ABA. Both compounds are known to turn
down the activity of the PM H⫹-ATPase (Beffagna et
al., 1995; Goh et al., 1996; Lino et al., 1998).
Could it be that the PM H⫹-ATPases, the endogenous current, and auxin have a role in growth and
differentiation of the cambial region? We think they
have. During growth and differentiation of cambial
cells, K⫹ ions are taken up, the surface area of the
cells expands, and cell wall polysaccharides/proteins
are secreted (Savidge, 1996). In spring, high concentrations of endogenous auxin are present in young
shoots and twigs (Little and Savidge, 1987), and a
promotion of cambial activity by auxin that was applied to debudded stems has been demonstrated
(Wareing et al., 1964). This auxin was transported to
the cambium and its young derivatives (Lachaud and
Bonnemain, 1984; Tuominen et al., 1997; Uggla et al.,
1998). Auxin has also been shown to mediate an
increase in H⫹-ATPase transcripts in the plasma
membrane of maize coleoptiles (Hager et al., 1991;
Frias et al., 1996), and small “auxin up RNA’s” and
“auxin genes” have been found during cell elongation (Abel et al., 1994; Abel and Theologis, 1996).
Similar effects of auxin are likely to occur in the
cambial region of poplar plants because the number
of PM H⫹-ATPases and the ionic current density
increased with the addition of auxin. The increase in
current density indicates more H⫹ ions in the apoplast and hyperpolarization of the membrane potential in cells of the cambial zone. An elevated proton
gradient and a higher membrane voltage favor the
uptake of sugars via symport with protons into the
rapidly growing cambial cells and an influx of K⫹
ions due to opening of ion channels in the plasma
membrane of expanding young vessel cells (Briskin
and Gawienowski, 1996). We were recently able to
demonstrate that the formation of fewer and larger
vacuoles during cambial reactivation is caused by
increased K⫹ uptake, probably modulated by the
Figure 10. Typical examples of short-term effects of IAA and ABA on the endogenous ionic
current of shoot cuttings from P. nigra. A, IAA
(30 ␮M) was added for 2 h to the artificial pond
water (APW) bathing a dormant shoot cutting in
December. B, ABA (30 ␮M) was added for 2 h to
the APW bath of a shoot cutting during high
cambial activity in April. Absolute current densities are given and projected onto the cross
sections of the shoots as columns of inward or
outward current.
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 129, 2002
Plasma Membrane H⫹-ATPase in Poplar
activity of a PM H⫹-ATPase (Arend and Fromm,
2000). Thus, the auxin-induced up-regulation and
activation of a PM H⫹-ATPase in poplar plant cells
seems to be a prerequisite for nutrient uptake and
growth of cambial and young xylem cells. This is
similar to roots, where inward H⫹-current is correlated with nutrient uptake and plasmatic growth at
the root apex, and outward current with uptake of
minerals and cell enlargement in the elongation zone
(Weisenseel et al., 1992). A change in current direction during the season, as for instance measured in
the cambial zone, may, therefore, be an indicator of a
shift in the mode of growth. ABA, in contrast to
auxin, strongly reduced the endogenous current of
poplar plants. This correlates positively with the
strong inhibitory effect of ABA on wood growth
(Fromm, 1997). In late summer, the formation of latewood has also been attributed to an increase in the
level of endogenous ABA in the cambial zone, effecting cessation of cambial activity (Jenkins and Shephard, 1974; Wodzicki and Wodzicki, 1980). Our
present results indicate a role for auxin in hardwood
formation of poplar trees via activation of a PM
H⫹-ATPase in the cambial region.
MATERIALS AND METHODS
Plant Material and Growth Conditions
Poplar (Populus nigra var italica L. and Populus trichocarpa Torr. et Gray.)
plants were selected for the experiments because of the well-known advantages of the genus Populus as a model for hardwood secondary vascular
growth (Chaffey, 1999). For electron microscopic investigations (SEMenergy-dispersive x-ray analysis), immunoblot analysis, and immunocytochemical procedures, twig tissue samples were collected from mature poplar (P. trichocarpa) trees grown in a forest near the campus of the Technical
University of Munich. For measurements of endogenous current, stem
cuttings with a length of approximately 50 mm and a diameter of 5 to 7 mm
were taken from 1- to 2-year-old upright shoots sprouting from the trunk or
roots of poplar trees (P. nigra) growing on the campus of the University of
Karlsruhe.
SEM and X-Ray Microanalysis
Small sections of twig tissue were cut with a razor blade and immediately
shock-frozen in liquid isopentane at its melting point. After freeze-drying,
the samples were coated with chromium and examined in a scanning
electron microscope (AMR 1200, Leitz, Wetzlar, Germany) fitted with a
KEVEX 4000 x-ray analyzer. Element-specific x-ray spectra were obtained
from a reduced scan raster area at 1,000⫻ magnification. Relative potassium
concentrations were expressed as peak to background ratio from 10 recorded spectra. For visualizing the distribution of potassium in the twig
tissue, potassium-specific x-ray signals were recorded using the elementspecific scan modus of the microscope.
Antibodies against Plasma Membrane Hⴙ-ATPase
The monoclonal antibody 46 E5 B11 was chosen for western blotting and
immunolocalization, because of its strong immunoreactivity against PM
H⫹-ATPase (Lützelschwab, 1990; Villalba et al., 1991; Baur et al., 1996;
Langhans et al., 2001) The hybridoma clone 46 E5 B11 was used to produce
antibodies against a PM H⫹-ATPase of maize (Zea mays; Lützelschwab,
1990; Villalba, et al., 1991). For the experiments described here, subclone 46
E5 B11 F6 was employed according to Baur et al. (1996). The specificity of
this antibody against a PM H⫹-ATPase of poplar plants was tested by
western-blot analysis (see below). For comparison, the polyclonal antibody
Plant Physiol. Vol. 129, 2002
L3E raised against a polypeptide corresponding to the PMA 2 isogene
(plasma membrane H⫹-ATPase 2) of Nicotiana plumbaginifolia were used for
western blotting and immunolocalization (Morsomme et al., 1998).
SDS-Page and Immunoblot Analysis
Poplar twigs were collected in spring during cambial activity and in
autumn during cambial dormancy. From each sample, 0.5 g of fresh tissue
was homogenized in an ice-cooled mortar with 5 mL of 10 mm Tris-HCl, 5
mm EDTA, and 1% (v/v) plant protease inhibitor cocktail (Sigma, St. Louis),
pH 6.8, and filtered through a layer of gauze. The filtrate was centrifuged at
20,000g for 10 min at 4°C, and the resulting pellet was resuspended in 2 mL
of solubilization buffer containing 50 mm Tris-HCl, 2% (w/v) SDS, and 3%
(v/v) ␤-mercaptoethanol, pH 6.8. After incubation for 1 h at 25°C, the
solution was centrifuged for 1 min at 8,000g, and a sample of the supernatant was loaded without heating onto an 8% (v/v) SDS-polyacrylamide
minigel. Protein standards of known Mr (prestained broad range, Bio-Rad,
Hercules, CA) were run on the same gel. Proteins were transferred to a
polyvinylidene difluoride membrane using a semidry transfer unit (TRANSBLOT SD, Bio-Rad). The membrane was washed in Tris-buffered saline
(TBS), blocked for 2 h with 1% (w/v) bovine serum albumin (BSA) in TBS,
incubated for 1 h with the monoclonal mouse antibody 46 E5 B11 F6, diluted
1:500 or with the polyclonal rabbit antibody L3E, and diluted 1:1,000 both in
TBS containing 1% (w/v) BSA. After washing in TBS containing 0.05% (v/v)
Tween 20, the membrane was incubated for 1 h with 1-nm gold-conjugated
goat anti-mouse antibody or anti-rabbit antibody (British Biocell International, Cardiff, UK), diluted 1:400 in TBS containing 1% (w/v) BSA. The
labeling of the membrane was developed with a silver enhancing Kit (British
Biocell International) according to the manufacturer’s instructions.
Fluorescence Microscopy Immunolabeling
Samples of 10 to 20 mm in length were cut from 1- or 2-year-old twigs
and fixed with 3% (w/v) formaldehyde (freshly prepared from paraformaldehyde) and 3 mm EGTA in 50 mm PIPES, pH 6.4, for 1 h at room
temperature. After washing in PIPES solution, cross sections of 50 ␮m
thickness were cut with a microtome and rinsed in PBS, pH 7.2. To reduce
unspecific labeling, the sections were blocked with 100 mm glycine in PBS-T
(PBS containing 0.2% [w/v] Tween 20) and 3% (w/v) BSA in PBS-T, both for
30 min. For immunolocalization, sections were incubated for 2 h at 37°C
with the monoclonal mouse antibody 46 E5 B11 F6 diluted 1:500 or with the
polyclonal rabbit antibody L3E diluted 1:1,000, both in PBS containing 0.5%
(w/v) BSA. After washing in PBS, the sections were incubated for 1 h at
37°C with Cy3 labeled anti-mouse antibody, respectively, anti-rabbit antibody (Cy3 conjugates, Dianova, Hamburg, Germany) diluted 1:250 in PBS
containing 0.5% (w/v) BSA. After the incubation period, the sections were
rinsed in PBS-T and then viewed using an axiophot microscope (Zeiss, Jena,
Germany) equipped with the filter combination 546-nm exciter and 590-nm
emitter. Photographs were taken with Kodak Ektrachrome 320 T film (Eastman Kodak, Rochester, NY). Incubations in medium containing Cy3conjugated secondary antibody without primary antibody or in medium
with mouse nonimmune IgG diluted 1:200 instead of primary antibody
served as controls.
Electron Microscopy Immunolabeling
Small cuttings from poplar twigs were collected in spring after cambial
reactivation and fixed for 1 h at room temperature with 3% (w/v) formaldehyde (freshly prepared from paraformaldehyde) in PBS, pH 7.2, containing 1% (w/v) saponin for permeabilization of cells. After washing in PBS-T,
unspecific binding sites were blocked for 30 min with 100 mm glycin and for
another 30 min with 5% (v/v) goat normal serum and 1% (w/v) BSA, both
in PBS-T. For immunolabeling, samples were incubated overnight at 4°C
with the antibody 46 E5 B11 F6 diluted 1:500 in PBS containing 0.5% (w/v)
BSA, then washed in PBS-T and incubated for 2 h at room temperature with
1 nm gold-conjugated goat anti-mouse antibody (British Biocell International) diluted 1:400 in PBS containing 0.5% (w/v) BSA. Controls were
incubated with nonimmune mouse IgG in place of the specific primary
antibody. After washing in PBS-T, the samples were post-fixed for 30 min
with 2% (v/v) glutaraldehyde in PBS and washed in distilled water. For
visualizing the 1-nm gold particles at the electron microscope level, silver
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
1661
Arend et al.
enhancement was carried out with the silver-enhancing kit from British
Biocell International according to the manufacturer’s instructions before
the samples were dehydrated and embedded in Spurr’s epoxy resin. This
unusual procedure greatly improved the detection of a PM H⫹-ATPase.
Ultrathin sections from the peripheral tissue layers were cut with a diamond knife on an ultramicrotome (LKB, Uppsala), transferred onto Formvar coated copper grids, and stained with lead citrate. Sections were
examined using a Zeiss EM 10 transmission electron microscope operated
at 80 kV.
Measurement of Endogenous Ionic Currents
Stem cuttings of approximately 50 mm in length from shoots were
trimmed at their apical surface to an angle of 30° and fastened vertically into
small petri dishes with a front window of cover glass and a downward
extending tube of Plexiglas. The dishes were filled with 25 mL of APW
containing 1.0 mm NaCl, 0.1 mm KCl, 0.1 mm CaCl2, and 1.0 mm MES,
adjusted to pH 6.0 with Tris. The dishes were suspended into the stage of an
inverted microscope and viewed from the front with a horizontal stereoscope (Leitz). The cuttings were left to settle and recover from handling for
30 to 60 min before the measurements. Endogenous current was measured
with a three-dimensional vibrating-probe set-up as described by Weisenseel
et al. (1992). In brief, the three-dimensional probe measures the total current
density and its spatial components, i.e. it measures current density vectors.
For the measurements, a 50°-tilted, insulated metal electrode (SS300305A,
Micro Probe, Clarksburg, MD) with a 30-␮m spherical tip of platinum black
was vibrated lengthwise and approximately 200 ␮m above the green cortex
parenchyma, the cambial zone, young and mature xylem cells, and the pith
parenchyma. Three consecutive measurements were carried out at each site
by rotating the probe, which was mounted to the bottom of a horizontally
revolving stage, into three positions separated by 30° and 90°, respectively.
The probe was vibrated with a frequency of 317 Hz, an amplitude of 35 ␮m,
and a duration of 20 s in each position. At sites with an electric potential
difference, originating from ionic current flow through the aqueous medium, this voltage was picked up by the electrode and transformed into an
AC voltage by the vibration. The latter was measured using a lock-in
amplifier (5210, PerkinElmer Life Sciences, Boston). The three values of
voltage measured at each site were then used to calculate the local current
density vector with the aid of a custom-made algorithm. Current densities
may be translated into ion fluxes by equating 1 ␮A cm⫺2 with 10 pmol cm⫺2
s⫺1 of monovalent ions.
ACKNOWLEDGMENTS
We thank Dr. Martin Lützelschwab for kindly providing the antibody
clone 46 E5 B11 F6, Dr. Marc Boutry for polyclonal antibodies, and Barbara
Schlicke for technical assistance.
Received February 8, 2002; returned for revision March 12, 2002; accepted
May 3, 2002.
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