Journal of Experimental Botany, Vol. 55, No. 403, pp. 1635–1641, August 2004
DOI: 10.1093/jxb/erh193 Advance Access publication 2 July, 2004
RESEARCH PAPER
Lateral ABA transport in maize roots (Zea mays):
visualization by immunolocalization
Daniela Schraut1, Cornelia I. Ullrich2 and Wolfram Hartung1,*
1
Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, Julius-von-Sachs-Platz 2,
97082 Würzburg, Germany
2
Institut für Botanik, Technische Universität, Schnittspahnstr. 3, 64287 Darmstadt, Germany
Received 14 January 2004; Accepted 5 May 2004
Abstract
The intensity of an ABA (abscisic acid) signal as a rootto-shoot signal, as well as its action on root hydraulic
conductivity, strongly depends on the distribution of
ABA during its radial transport across roots. Therefore
ABA was visualized by immunolocalization with monoclonal ABA antibodies under conditions of lateral water
flow induced by the application of a pressure gradient
to the cut surface of the mesocotyl of maize seedlings.
From the labelling of rhizodermis, hypodermis, cortical
cells, and endodermis of roots of hydroponically (no
exodermis) and aeroponically (with exodermis) grown
seedlings it is concluded that the exodermis acts as
a barrier to apoplastic transport that controls ABA
uptake and efflux, but that the endodermis can easily
be overcome via an apoplastic bypass. In longitudinal
sections the strongest ABA signals originated from
the root cap and the meristematic root tip, which is in
agreement with the non-vacuolated cells of these
tissues being an effective anion trap for ABA.
Key words: ABA immunolocalization, exodermis, lateral ABA
transport, maize root sections.
Introduction
The role of abscisic acid as a long-distance stress signal has
now been well established. When the soil is getting dryer
ABA is synthesized within the roots, released to the xylem,
and transported to the leaf blades where transpiration is
restricted and to the leaf meristems, where leaf development is adapted to the environmental stress conditions
(Sauter et al., 2001). In addition to its synthesis, ABA may
be of external origin, as released from other plant roots
and from ABA-producing micro-organisms such as soil
fungi. Under natural conditions, ABA and its conjugates
were shown to be present in the soil solution of moist soils
at concentrations up to 30 nM (Hartung et al., 1996; Sauter
and Hartung, 2000).
The intensity of the ABA stress signal to the stem must
also be regulated during lateral transport through the roots.
Assuming that ABA is transported within the symplast,
changes of water flow, mainly due to varying transpiration,
will dilute or concentrate ABAxyl. An apoplastic bypass
flow of ABA as shown by Freundl et al. (1998) could
compensate for the dilutions caused by increased transpiration. The Casparian bands of the endodermis could form
a first barrier to ABA transport. Once a second apoplastic
barrier, the Casparian bands in the hypodermis have been
formed, the uptake and lateral transport of external ABA
are also affected (Hartung et al., 2002).
The aim of this study was to show lateral transport of
ABA and its possible regulation in maize roots. Immunolocalization of ABA in cross-sections of maize roots, grown
hydroponically (without exodermis), and aeroponically
(with exodermis) was used to visualize the distribution of
ABA under transpiring and non-transpiring conditions. For
cytokinins and auxin in plant tumours, immunolocalization
with monoclonal antibodies has been established by
Veselov et al. (2003). Their technique was adapted and
optimized for ABA detection in root tissues. Until now, few
attempts have been undertaken to study ABA distribution
in plant tissues by immunolocalization. Sossountzov et al.
(1985) and Pastor et al. (1995) used polyclonal ABA
antibodies for immunogold and immunofluorescence experiments to study the ABA compartmentation in green
plant cells. Wächter et al. (2003) recently showed ABA
* To whom correspondence should be addressed. Fax: +49 931 888 6158. E-mail: [email protected]
Journal of Experimental Botany, Vol. 55, No. 403, ª Society for Experimental Biology 2004; all rights reserved
1636 Schraut et al.
accumulation in xylem parenchyma cells in plant tumours
and stems. The present study describes for the first time
ABA distribution in roots during its radial transport as
induced by the application of a pressure gradient to the cut
surface of the mesocotyl, thus imitating the suction force of
the xylem. By applying this technique it was possible to
demonstrate that a significant portion of external ABA from
the soil is transported from the cortex directly across the
endodermis into the metaxylem vessels and that the exodermis can prevent ABA loss into the rhizosphere.
Materials and methods
Plant material
Seeds of maize (Zea mays L. cv. Helix, Kleinwanzlebener Saatzucht
AG, Einbeck, Germany) were germinated on filter paper soaked with
0.5 mM CaSO4 at 26 8C in the dark for 4 d. Seedlings were transferred
to aerated hydroponic or to aeroponic culture and kept in a greenhouse
with an additional light source (mercury vapour lamp, 200 lmol mÿ2
sÿ1; day/night 16/8 h, 25/17 8C) for 7 d. The standard nutrient
solution used was modified from that of Pirson and Seidel (1950) and
contained the following nutrients: 1.5 mM KH2PO4, 2.0 mM KNO3,
1.0 mM CaCl2, 1.0 mM MgSO4, 18 lM FeNaEDTA, 8.1 lM H3BO3,
and 1.5 lM MnCl2 at pH 5.5. The conditions of hydroponic and
aeroponic culture were previously described in detail (Freundl et al.,
1998).
Immunolocalization of ABA
For immunolocalization studies 11-d-old Zea mays seedlings, grown
either hydroponically or aeroponically, were used. The average
length of the hydroponically grown roots was 32 cm, that of the
aeroponically grown roots 44 cm. The total root surface area of the
root system of the seedlings, measured with a computer-assisted
system (scanwise 1.2.0.5, Agfa; WinRHIZO Pro V.2002b, Regent
Instruments Inc.) was 87 cm2 and 120 cm2, respectively. A radial
water flow was established either by transpiration in intact seedlings
kept in the light or by applying a suction pressure of ÿ0.06 MPa to
the cut surface of excised root systems at the level of the mesocotyl
emerging in an aerated nutrient solution, as described in detail by
Freundl et al. (1998). In some of the experiments 100 nM (+)-cistrans-ABA (Sigma Chemicals, Deisenhofen, Germany) was added to
the medium surrounding the roots. This ABA concentration was used
for the following reasons: (i) it can be expected in the apoplast of root
tissues of stressed plants (Slovik et al., 1995); (ii) in the plants of this
study it causes an increase of ABAxyl of up to 30 nM, which is in
the range of moderately stressed maize plants in natural conditions
(Sauter and Hartung, 2000); and (iii) this ABA concentration
increases the water conductivity of maize roots substantially (Hose
et al., 2000).
Tissue preparation
In order to immobilize ABA immediately by covalent binding to
proteins after a suction experiment, root segments of different zones
were fixed for at least 24 h with 3% (w/v) paraformaldehyde in 4%
(w/v) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) containing 0.1% (v/v) Triton X-100 at 4 8C. After washing the fixed
samples with stabilizing buffer (SB; 50 mM HEPES, 5 mM MgSO4,
5 mM EGTA, pH 6.9) and PBS (phosphate buffered saline: 137 mM
NaCl, 2.7 mM KCl, 7.9 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.3) to
remove the fixative, the samples were dehydrated with a graded series
of ethanol/PBS. Tissues were then infiltrated with increasing con-
centrations of Steedman’s wax (a polyester with low melting point;
polyethyleneglycol-distearate in 1-hexadecanol 9:1 (w/w); Vitha
et al., 1997). Sections of 11 lm were prepared with a sliding
microtome (HN 40, Jung, Heidelberg, Germany) and collected on
poly-L-lysine-coated slides.
Immunocytochemistry
Sections were dewaxed in a series of ethanol from 30% to 100% in
20% intervals (no technical grade ethanol because of increased
autofluorescence) and rinsed with SB, methanol (ÿ20 8C) and PBS/
1% Tween 20 (v/v). Before incubating overnight with the primary
monoclonal ABA antibody (ABA-15-I-c-5; Agdia/Linaris, WertheimBettingen, Germany), the sections were pretreated with rabbit
serum for 1 h to reduce unspecific binding. The mouse monoclonal
hybridoma antibody, raised against ABA–BSA conjugates, is highly
specific to (+)-cis-trans-ABA (Weiler, 1982). After washing with
PBS/Tween 20 the sections were incubated with the red Alexa
conjugate 568 (568 goat anti-mouse IgG, H+L, Molecular Probes,
Göttingen, Germany; excitation 568 nm, emission 598–608 nm) as
a secondary antibody for 2 h. After washing with PBS and staining
with toluidine blue (to quench the autofluorescence of the lignified
cell walls; Peterson, 1988) and with aniline blue, sections were
embedded in 1,4-diazabicyclo-(2,2,2)octane (DABCO; 25 mg mlÿ1
in PBS/glycerol 1+9). The covered slides were sealed with nail
varnish. Sections were viewed with a confocal laser scanning
microscope (CLSM; Zeiss LSM 5 Pascal 5, Axioskop 2 mot plus),
excitation 543, HFT 543, NFT 635, emission (BP) 560–615). Images
were converted to a false colour glow mode to improve the contrasts. In
Figs 1i and 2h (see Results) the vertical bar shows a spectrum of
colours from black (no signal) to white (strongest signal) indicating the
intensity of fluorescence originating from the secondary antibody,
Alexa 568-conjugate, and thus the increasing ABA concentrations.
From the intensity of the signals absolute ABA concentrations cannot
be concluded. However, ABA-transport experiments performed with
the same system under comparable conditions (Freundl et al., 1998,
2000), as well as computer simulations about compartmental ABA
concentrations in root cell compartments (Slovik et al., 1995) indicate
the fluctuation range of ABA concentration. Several controls were
included in order to show the specificity of the method. Some sections
were treated with neither ABA antibody nor Alexa, others were
treated with ABA antibody or with Alexa separately, others with
ABA antibody presaturated with an excess of ABA (500 nM) and with
Alexa. The experiments were performed 5–8 times with 60–80 tissue
sections each.
Results
The validity of ABA-immunolocalization
Although the monoclonal ABA antibody used in this study
is highly specific to (+)-cis-trans-ABA (Mertens et al.,
1985) careful controls were essential to avoid artefacts and
misinterpretations. Autofluorescence of the cell walls,
especially of the rhizodermis, exodermis, and xylem elements may interfere (Fig. 1k). Therefore, toluidine blue was
used to suppress autofluorescence (Fig. 1i, l, m, n), according to Peterson (1988). The control shown in Fig 1i, which
was not incubated with the primary and secondary antibodies, was treated with toluidine blue. It did not show
ABA signals. In addition, controls without the primary
antibody (Fig. 1l) or the secondary antibody (Fig. 1m) did
not emit fluorescence signals. When sections were treated
Immunolocalization of ABA
with a monoclonal ABA antibody that had been preincubated with an excess of ABA (500 nM) no ABA signals
were visible either (Fig. 1n). It is therefore concluded that
fluorescence of the sections in Fig. 1a–i, l–n exclusively
originate from ABA.
ABA in hydroponically grown maize roots
When ABA was absorbed from a water flow containing
100 nM ABA, created by a ÿ0.06 MPa pressure gradient, the
rhizodermis, hypodermis, endodermis, and the early metaxylem vessels with their surrounding xylem parenchyma
cells showed strong ABA signals (Fig. 1b), while cortical
cells and the late metaxylem vessels were extremely weakly
labelled. In the rhizodermis and hypodermis, the radial cell
walls exhibited the strongest label, together with the early
metaxylem and protoxylem vessels in the stele.
When seedlings were grown without their caryopses,
stronger ABA signals appeared in the xylem parenchyma
cells and also in the cortical cells (Fig. 1c). When a radial
water flow was created by leaf transpiration in intact, nondecapitated, seedlings (Fig. 1e), similar ABA signals were
obtained to those under the artificial pressure gradient
(Fig. 1b). By comparison, weak signals originated from
endogenous ABA (Fig. 1g): only the rhizodermis and
hypodermis were labelled.
In cross-sections between the root tip and the root hair
zone (17–18 mm behind the root tip, see Fig. 2a, arrow),
ABA signals were more homogeneously distributed over
the sections and were more intensive than in the root hair
zone (Fig. 2b, d).
ABA in aeroponically grown roots
When maize seedlings were grown aeroponically (Fig. 1d),
i.e. under more natural conditions, Casparian bands were
formed in the hypodermis. This barrier was shown to
reduce uptake of external ABA and to prevent the release of
internal ABA (Hose et al., 2000, 2001). Different from the
endodermis, these barrier properties became evident by the
strong labelling of the outer tangential cell walls of the
exodermis and the rhizodermis, whereas the inner tangential walls of the exodermis only emitted weak signals (Fig.
2h). This was also obvious in the fluorescence intensity
profiles of Fig. 3. In addition, the radial walls of the
endodermis exhibited a particularly strong fluorescence
(Fig. 2f, g). Together with the weak label of the inner and
outer tangential walls of the endodermis, this is consistent
with an apoplastic flow of the lipophilic ABA directly
across the Casparian bands of the endodermis. Signals from
endogenous ABA resembled those of hydroponically
grown roots (Fig. 1h).
ABA in longitudinal sections
In longitudinal sections of maize seedlings, the strongest
signals were emitted from the root cap and the meriste-
1637
matic root tip (Fig. 2b). Here the whole cells were
labelled due to scarce vacuolization of the meristematic
cells. The more basal regions fluoresce much less. From
5.9 mm onwards, stelar cells exhibited significant signals
(Fig. 2c). The longitudinal sections indicate that the root
tip apically from the root hair zone seems to be less
important for radial and longitudinal basipetal ABA
transport, although weak fluorescence in the intermediate
zone could indicate that ABA is transported but not
accumulated.
Discussion
In the work presented here the distribution of ABA in the
presence of a radial water flow was visualized for the first
time. Besides endogenously synthesized ABA, roots also
absorb external ABA produced by soil micro-organisms
and released from other plant roots (Hartung et al., 1996).
External ABA passes the rhizodermis and the cortical cells
predominantly along the lateral cell walls. In all experiments, the strongest ABA-specific signals originated from
the cell walls together with the cytoplasmic layer. Vacuoles
were not visibly labelled because the vacuolar ABA concentration is lower than the cytosolic by at least a factor
of 10 (Kaiser et al., 1985) and because in the vacuoles the
protein content, to which ABA could be bound with EDAC,
is too low. When cells were not or only weakly vacuolized
almost the whole cells were strongly labelled, as obvious
from longitudinal (Fig. 2b) and cross-sections (Fig. 2d)
obtained from 1.7–1.8 mm behind the tip. In this zone of the
maize roots increased ABA concentrations maintain the
growth of the root tip under conditions of water deficiency
(Saab et al., 1990; Sharp et al., 1990). Whether ABA is
exclusively transported in the cell walls or in the adjacent
cytoplasmic layer as well cannot be recognized from the
images presented here. In the endodermis again the radial
cell walls were strongly labelled, including the Casparian
bands. The inner and outer tangential walls showed much
weaker signals. This is consistent with a direct ABA
transport across the radial endodermal cell walls with an
apoplastic bypass flow of water. After passing the endodermis, the early metaxylem vessels are rapidly reached.
The early metaxylem vessels of maize roots in and slightly
above the root hair zone were found to be in a developing
stage and to conduct water at very low rates (Aubin et al.,
1986). Their cytoplasmic layer will accumulate ABA
arriving with the water flow before it is released into the
functioning vessels and thus into the transpiration stream.
During its radial transport through the root, ABA also
increases the hydraulic conductivity of the root. In the
experiments shown here 100 nM ABA stimulated the radial
water flow 2-fold, from 2.3310ÿ9 to 4.6310ÿ9 m3 mÿ2 sÿ1
(D Schraut, unpublished results). From the present results
(Figs 1, 2) it can be concluded that the site of ABA action
may be in the xylem parenchyma cells (XP) that surround
1638 Schraut et al.
Fig. 1. Immunocytochemical localization of ABA in cross-sections of maize roots. (a–c, e–n) Hydroponically grown; (d, h) aeroponically grown. (a)
Section viewed by light microscopy showing rhizodermis (RH), hypodermis (HY), endodermis (EN), and the stele with early (EM) and late (LM)
metaxylem vessels. (b–n) Sections viewed by CLSM. (b–h) External ABA (100 nM) added to the medium. Sections were incubated with monoclonal
ABA antibodies and with the secondary Alexa 568 antibodies and stained with aniline blue and toluidine blue. (b–d) Radial water flow was induced by
application of a pressure gradient of ÿ0.06 MPa. (b) Strong fluorescence signals in the rhizodermis (RH), hypodermis (HY), endodermis (EN), and early
metaxylem vessels (EM) in roots of hydroponically grown seedlings. (c) Roots of hydroponically grown plants after removing the caryopsis from the 4d-old seedling with stronger signals than in (b). (d) Roots of aeroponically grown seedlings with strong ABA labelling of rhizodermis and the outer
tangential cell wall of the exodermis with Casparian bands. The endodermis and early metaxylem vessels, but not the late metaxylem vessels, emitted
strong ABA signals. (e) Fluorescence signals of cross-sections of roots of intact seedlings, transpiring in the light. (f) Section of roots from excised root
systems. No pressure gradient was applied to the cut surface. Cells of rhizodermis, hypodermis, metaxylem, and, particularly, the endodermis, only
exhibited weak signals. (g–h) In the absence of external ABA very weak signals in rhizodermis and hypodermis, and extremely weak signals in
endodermis and early metaxylem vessels of hydroponically (g) and aeroponically (h) grown maize seedling roots can be seen. (i–n) Controls of
hydroponically grown seedlings treated with additional ABA (100 nM) during radial water flow. (i) Section incubated neither with the primary ABA nor
with the secondary Alexa 568 antibodies, but stained with aniline blue and toluidine blue. (k) Same conditions as (i) but not stained with toluidine blue.
Note the strong autofluorescence of the cell walls of root cells. (l) Section treated with primary ABA antibodies or (m) with Alexa 568-conjugates only.
(n) Section incubated with the primary ABA antibodies presaturated with ABA (500 nM). The vertical bar with a range of colours (dark red to white)
represents the increase of relative intensity of the ABA signals. Straight lines in (b), (d) and (n) indicate CLSM fluorescence intensity measurements (see
Fig. 3). Scale bars: 100 lm.
Immunolocalization of ABA
1639
Fig. 2. Immunocytochemical labelling of longitudinal and cross-sections of hydroponically and aeroponically grown maize roots. (a–d) In the presence
of external ABA (100 nM). (a) Longitudinal section of a root tip viewed by light microscopy, stained with aniline blue and toluidine blue. RT (root tip),
CAL (calyptra). (b, c) Longitudinal sections of roots 0–10 mm (b) and 50–100 mm (c) behind the tip with strong fluorescence signals of calyptra,
rhizodermis, and hypodermis 70–80 mm behind the root tip. (d) Signals of stelar tissues in a cross-section 1.7–1.8 mm behind the tip with signals
homogeneously distributed over the whole section. (e–g) Endodermis in cross-sections 6–8 cm behind the root tip. (e) Endodermis of hydroponically
grown roots with Casparian bands (CB). (h) Rhizodermis and exodermis in a section of aeroponically grown roots. The vertical bar with a range of
colours (dark red to white) represents the increase of the relative intensity of the ABA signals. Scale bars: 1 mm (a), 100 lm (d), 200 lm (b, c), and 50 lm
(e–h).
the early and the developing metaxylem vessels. Water flux
into roots and its flow into shoots is known to be dependent
on both osmotic forces and transpiration. ABA apparently
controls ion fluxes as well as water movement. Although
the exact site of ABA action is still obscure, XP cells in
the stele are very likely targets. Such XP cells in roots
have been shown to be highly active in ion release into the
xylem vessels (Läuchli et al., 1971). More recently
voltage-dependent XP-located channels were found to
respond to water stress, and ABA was found to inhibit K+
release from XP into the vessels through the outward
rectifying K+ channels SKOR (Roberts, 1998; Gaymard
et al., 1998; Roberts and Snowman, 2000). The assumption
that ABA also controls water channels (Wan et al., 2004)
would be in agreement with the findings of Otto and
Kaldenhoff (2000), about a strong gene expression of water
channels in the stele of tobacco. By contrast, Hose (2001)
detected a higher gene expression of different water
channels in the cortex of maize than in the stele. Assuming
that those aquaporins are under ABA control, it is suggested that here the hypodermis and exodermis, including the
adjacent cortical cells, are the best candidates for additional
sites of ABA action. This applies particularly to roots of
aeroponically grown seedlings.
The radial ABA transport is diminished in the aeroponically grown roots when the hypodermis exhibits Casparian
bands. Freundl et al. (2000) and Hose et al. (2000) showed
that the exodermis is a barrier to radial ABA transport, for
both efflux and influx. Barrier properties must exist in tissue
zones with strong fluorescence, i.e. the rhizodermis and the
outer tangential walls of the exodermis (Figs 1d, 2g). In
these zones ABA could also accumulate at such barriers.
The signals from the inner tangential exodermal walls were
much weaker (Fig. 3b). The labelling of the endodermis
showed undisturbed flux of ABA directly into the metaxylem vessels. Different from the hydroponically grown
roots, cortical cells and the living stelar cells of aeroponically grown roots exhibited stronger fluorescence. This may
200
Cortex
a
Metaxylem
E n d o d er m i s
300
Hypodermis
Rhizodermis
rel. fluorescence intensity
1640 Schraut et al.
Stele
100
0
100
150
200
250
300
350
400
450
Cortex
b
Metaxylem
200
Endodermis
rel. fluorescence intensity
300
Exodermis
Rhizodermis
distance [µm]
Stele
100
0
-50
0
50
100
150
200
250
300
350
400
450
Rhizodermis
rel. fluorescence intensity
distance [µm]
300
200
c
Cortex
Stele
100
0
0
50
200
Cortex
Stele
Metaxylem
300
Metaxylem
Rhizodermis
+
Hypodermis
rel. fluorescence intensity
distance [µm]
d
Cortex
100
0
-50
50
150
250
350
450
distance [µm]
Fig. 3. CLSM fluorescence intensity profiles of Alexa Fluor 568-labelled monoclonal ABA antibodies along the straight lines indicated in Fig. 1b, d, n
and Fig. 2d. Profiles across sections of hydroponically (a, c, d) and aeroponically (b) grown roots. (c) Fluorescence profile of a cross-section 1.8 mm
behind the tip and (d) of a control where the section was incubated with ABA antibodies presaturated with 500 nM ABA.
be a result of the weak leakage of internal and external
ABA to the medium, caused by the Casparian bands of the
exodermis.
In conclusion, cross-sections and longitudinal sections of
maize seedling roots, together with carefully performed control experiments, can be used to visualize the radial transport
pathways of ABA. These data are consistent with earlier
findings with ABA-flux analyses on the barrier properties of
the exo- and endodermis.
Acknowledgements
We are grateful to Dr Markus Langhans (TU Darmstadt) for his
valuable advice with immunostaining, to Professor EW Weiler
Immunolocalization of ABA
(University Bochum) for the generous supply of ABA antibodies, to
Robin Wacker (University Würzburg) for help with the microtome,
to Bianca Röger for skilful technical help, and to the Deutsche
Forschungsgemeinschaft (Ha 963-11/1) for financial support.
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