2925
Development 125, 2925-2934 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
DEV0176
Localized changes in apoplastic and cytoplasmic pH are associated with root
hair development in Arabidopsis thaliana
Tatiana N. Bibikova1, Tobias Jacob1,2, Ingo Dahse2 and Simon Gilroy1,*
1Department of Biology, The Pennsylvania State University, 208 Mueller Lab, University Park, PA 16802, USA
2Friedrich Schiller University, Institute of Biochemistry and Biophysics, Department of Biophysics, Philosophenweg
12, 07743
Jena, Germany
*Author for correspondence (e-mail: [email protected])
Accepted 14 May; published on WWW 9 July 1998
SUMMARY
Morphogenesis in plants is characterized by highly
regulated cell enlargement. However, the mechanisms
controlling and localizing regions of growth remain
essentially unknown. Root hair formation involves the
induction of a localized cell expansion in the lateral wall of
a root epidermal cell. This expanded region then enters a
second phase of localized growth called tip growth. Root
hair formation therefore provides a model in which to study
the cellular events involved in regulating localized growth
in plants. Confocal ratio imaging of the pH of the cell wall
revealed an acidification at the root hair initiation site. This
acidification was present from the first morphological
indications of localized growth, but not before, and was
maintained to the point where the process of root hair
initiation ceased and tip growth began. Preventing the wall
acidification with pH buffers arrested the initiation process
but growth resumed when the wall was returned to an
acidic pH. Cytoplasmic pH was found to be elevated from
approximately 7.3 to 7.7 at the initiation site, and this
elevation coincided with the acidification of the wall.
Preventing the localized increase in cytoplasmic pH with 10
mM butyrate however did not inhibit either the wall
acidification or the initiation process. In contrast, there was
no detectable gradient in pH associated with the apex of tip
growing root hairs, but both elevated apoplastic pH and
butyrate treatment irreversibly inhibited the tip growth
process. Thus the processes of tip growth and initiation of
root hairs show differences in their pH requirements. These
results highlight the role of localized control of apoplastic
pH in the control of cell architecture and morphogenesis in
plants.
INTRODUCTION
material is confined to the expanding tip of the developing hair,
leading to the elongated hair-like morphology (Schnepf, 1986).
Research on the tip growth of pollen tubes, algal rhizoids,
fungal hyphae and root hairs suggests that a localized, highly
focused gradient of cytoplasmic Ca2+ is a strong candidate for
the spatial information determining the direction of growth
(e.g. Bibikova et al., 1997; Felle and Hepler, 1997; HoldawayClarke et al., 1997; Malho and Trewavas, 1996; Wymer et al.,
1997). However, no localized increase in cytoplasmic [Ca2+]
could be observed to precede or predict the site of root hair
initiation. Similarly, although the Ca2+-channel blocker
verapamil leads to inhibition of tip growth, root hair initiation
is unaffected (Wymer et al., 1997). Thus localized changes in
cytoplasmic [Ca2+] seem intimately involved in tip growth, but
are less convincing as central determinants of the initiation
process.
Changes in both cytoplasmic and apoplastic pH are
alternative candidates for determinants of localized growth.
Acidification of the cytoplasm has been associated with the
localized growth of Pelvetia rhizoids (Kropf and Gibbon, 1994)
and Funaria protonemata (Demkiv et al., 1994), although in
Root hairs are projections from epidermal cells of the root that
have been proposed to play critical roles in water and nutrient
uptake, and in anchoring the plant in the soil (reviewed by
Ridge, 1995). Only certain cells (‘trichoblasts’; Leavitt, 1904)
within the root epidermis are capable of undergoing
differentiation into root hairs. The patterning of such
differentiation is highly regulated. Thus the trichoblasts of
Arabidopsis are limited to a series of 8 cell files precisely
located at the intersection of two cortical cells (e.g. Dolan et
al., 1993).
To produce a root hair, a trichoblast undergoes 2 genetically
distinct processes: root hair initiation and tip growth of the
initiated hair (Ridge, 1995). During initiation, a highly
localized bulge is produced in the outer surface of the
trichoblast (Leavitt, 1904). The production of this bulge
represents a transition from diffuse longitudinal growth of the
epidermal cell to highly localized and tightly regulated growth
from the side of the cell. Once initiated the root hair
commences tip growth where deposition of new cell wall
Key words: Arabidopsis thaliana, Confocal ratio imaging, Cell wall
pH, Cytosolic pH, Root hair, Trichoblast
2926 T. N. Bibikova and others
fungi, pollen tubes (Fricker et al., 1997; Parton et al., 1997)
and tip growing root hairs (Herrmann and Felle, 1995), no
correlation between apical growth and pH gradients could be
found. Wall acidification also occurs in cells showing diffuse
growth (Mulkey and Evans, 1981), where the low pH may
activate proteins involved in wall relaxation (McQueen-Mason
et al., 1992). Although such ‘acid-growth’ does not explain all
aspects of the sustained elongation of plant cells, the limited
wall extension associated with the initiation process of root
hairs may be consistent with some degree of acid-based wall
relaxation. Additional evidence for a role of apoplastic pH in
plant morphogenesis arises from the report that local
application of the expansin proteins thought to mediate acidbased wall relaxation causes bulging of the leaf primordia in
tomato (Flemming et al., 1997). This localized control of tissue
expansion may have been sufficient to trigger the
developmental program of the primordia and induce leaf
formation.
We therefore tested whether changes in cytoplasmic or wall
pH were associated with the localized growth events associated
with the initiation process of root hairs of Arabidopsis thaliana.
We report that cytoplasmic pH increases, and wall pH
decreases at the root hair initiation site. Preventing the change
in apoplastic, but not cytoplasmic pH, reversibly arrests the
process of root hair initiation. Although disruption of
cytoplasmic and apoplastic pH inhibited tip growth, no distinct
gradient in cytoplasmic pH could be detected associated with
the growing tip of these cells.
MATERIALS AND METHODS
Plant material and measurement of growth kinetics
Seedlings of Arabidopsis thaliana (Columbia ecotype) were grown as
previously described (Wymer et al., 1997). Plants were used 4-7 days
after planting, at which time the roots were 1-3 cm long. Growth
kinetics were monitored on a Nikon Diaphot 300 microscope (Nikon
inc., Melville, NY, USA) using a 40× dry, Nikon, 0.7 numerical
aperture objective and differential interference optics as described
previously (Wymer et al., 1997). Unless otherwise stated, chemicals
were obtained from Sigma Chemical Co. (St Louis, MO, USA).
Fluorescent dyes were obtained from Molecular Probes Inc. (Eugene,
OR, USA).
Ratiometric measurement of cell wall pH
Root hair wall pH was monitored by pseudo-ratiometric confocal
imaging of the fluorescence from the pH-sensitive dye NERF-Cl,
conjugated to a 10 kDa dextran, and the pH insensitive dye Texas
Red conjugated to a 10 kDa dextran (Taylor et al., 1996). Both
dextran-conjugated dyes were infiltrated into the epidermal cell walls
of the root by incubating the root in 100 µM of the dextran dyes for
30 minutes. Wall pH was then imaged on the stage of a Zeiss axiovert
microscope attached to a LSM410 laser scanning confocal
microscope (Carl Zeiss, Thornwood, NY, USA), using a 40×, 0.75
numerical aperture, dry objective. Pseudo-ratio images were
collected using excitation from the 488 and 543 nm lasers of the
confocal microscope and emission collected at 515-540 nm (NERF)
and >590 nm (Texas Red) using 488/543 nm primary and 560 nm
secondary dichroic mirrors and appropriate interference filters. Each
frame represents a single 8 second scan of the laser. Pseudo-ratio
images were constructed by dividing the pH-dependent fluorescence
from the NERF with the pH-independent fluorescence from Texas
Red using IPLabs Spectrum image analysis software (Signal
Analytics, Vienna, VA). After background subtraction,
autofluorescence and dark current (<10% of the lowest fluorescence
signals from these dyes) were subtracted from each image, the ratio
image was calculated and masked using a binary mask derived from
thresholding the Texas Red image to approximately 90% of the
difference between background and the minimum Texas Red signal.
For the NERF/Texas Red and for the 2′,7′-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF)/rhodamine ratio imaging of
cytoplasmic pH (described below) photobleaching represented <5%
per channel per scan for each ratio image. The pseudo-ratiometric
approach was required because confocal resolution of fluorescence
was needed in order to image pH in the cytoplasm and wall of the
initiation site, and a true ratiometric pH indicator compatible with
both the expected apoplastic and cytoplasmic pH ranges (pH 4-6 and
7-8 respectively) and with our confocal microscope system is
unavailable.
Measurement of cytoplasmic pH
Trichoblasts and atrichoblasts, approximately 100 µm apical from the
zone of hair initiation were simultaneously microinjected with
BCECF linked to a 10 kDa dextran and rhodamine linked to a 10 kDa
dextran, as described previously for calcium green/rhodamine dextran
injections (Bibikova et al., 1997; Wymer et al., 1997). As noted by
others (Brauer et al., 1995; Kossegarten et al., 1997) the free acid,
non-dextran conjugated, form of BCECF was accumulated in the
vacuoles of root hairs over 10-30 minutes.
Coverslips with the microinjected seedlings were then placed on
the stage of the confocal microscope and imaged as described above
for NERF/Texas Red using excitation from the 488 and 543 nm lasers
and the emission collected at 515-540 nm (BCECF) and >590 nm
(rhodamine). Each frame represents a single 8 second scan of the laser.
Pseudo-ratio images were then constructed by dividing the pHdependent fluorescence from the BCECF with the pH-independent
fluorescence from the rhodamine, as described previously for calcium
green/rhodamine imaging (Bibikova et al., 1997). This pseudoratiometric approach was found to provide a reliable measure of the
cytoplasmic ion levels and distributions in root hairs (Bibikova et al.,
1997). The dextran-conjugated indicators remained in the cytoplasm
of root hair cells for >8 hours (Bibikova et al., 1997; Wymer et al.,
1997; and data not shown). Results from pseudo-ratiometric analysis
of BCECF dextran/Texas Red dextran gave identical results to pseudoratiometric analysis with BCECF dextran/rhodamine dextran;
therefore only the BCECF/rhodamine data is presented. In vitro
calibration of the BCECF/rhodamine was performed using 10 µM
BCECF dextran/rhodamine dextran, 100 mM KCl, 1 mM MgCl2, 1
mM ATP, 10 mM HEPES buffered to pH 6-8. In situ calibration was
performed using 10 µg/ml nigericin and 100 mM extracellular KCl
(Fricker et al., 1997). However, in 80% of cells tested, the in situ
calibration failed to elicit a reliable, sustained change in cytoplasmic
pH. The reason for this variability is unknown. Therefore due to the
reproducibility of the in vitro calibration and the low numbers of
microinjected cells available for in situ calibration, the values for
cytoplasmic pH presented were calibrated according to the in vitro
calibration. When successful, the in situ calibration agreed with the
in vitro calibration by >90%. Despite such caveats about the
calibration procedure it is generally thought that the fluorescent pHsensitive dyes are accurately reflecting changes in pH (Fricker et al.,
1997). Tip growing root hairs proved more difficult to microinject
than initiating root hairs and due to the larger volume of their
cytoplasm required more dye to be injected to lead to a reliable signal
for ratio analysis. The maximal signal attainable from dextranconjugated microinjected dyes in the tip growing root hairs was 50%
of that from the initiating root hair. Injecting more dye led to a failure
to maintain tip growth. Thus the ratio images of tip growing cells also
appear more ‘noisy’ than those of initiating cells, but still accurately
reflect cytoplasmic pH.
Bright-field images were also taken at each experimental time point
using the transmission detector of the confocal microscope and the
633 nm He/Ne laser attenuated to 10% with neutral density filters.
pH and root hair initiation 2927
RESULTS
calibrated in situ and in vitro. Fig. 2A shows an in vitro
calibration performed using 10 µM NERF/Texas Red dextrans,
100 mM KCl, 1 mM MgCl2, 10 mM of appropriate pH buffer
(pH 4-5, dimethylglutarate (DMGA) or citrate; pH 5-6.5 MES;
pH >6.5 HEPES) buffered to a range of pHs from 3 to 6. In
situ calibration was made using dyes infiltrated into cell walls
of roots that had been killed by freeze thawing and the
metabolic activity in the cytoplasm inhibited by 1 hour
incubation in ice cold ethanol. The measurements from these
dead roots were made to assess the responsiveness of the dyes
in the wall environment isolated from potential effects of the
living root cell protoplast. For in vivo calibration, roots were
loaded with the dextran-conjugated dyes and extracellular pH
buffered using 50 mM of the appropriate pH buffer (Fig. 2B).
Below 50 mM MES, HEPES, citrate or DMGA buffer
concentration, the pseudo-ratio imaging indicated no
reproducible change in cell wall pH (data not shown).
However, Fig. 2B shows that at 50 mM pH buffer, wall pH
changed in parallel to the pH set by the extracellular buffer
system in the range 4.5 to 6.0. We attribute this requirement
for high buffer levels to the strong natural pH buffering in the
cell wall. Thus the NERF/Texas Red ratio was found to be
linearly dependent on pH in the range 3 to 6 in the in situ
calibration in walls of dead and living roots and in the in vitro
calibrations (Fig. 2A). These three methods of calibration all
showed an identical change in ratio of 0.27-0.29/pH unit over
the linear pH 3 to 6 range. The ratio values were insensitive to
Ca2+ (0-50 mM), K+ (0-200 mM) and Cl− (0-200 mM) in vitro
(data not shown).
When the root was incubated in pH buffer, all regions of
Imaging of wall pH in Arabidopsis roots
To test the role of cell wall pH in the root hair initiation process,
we imaged the fluorescence from the dextran-conjugated dyes,
NERF and Texas Red infiltrated into the trichoblast wall. We
then determined wall pH by pseudo-ratio imaging, monitoring
the pH-sensitive fluorescence of NERF and comparing this to
the pH insensitive fluorescence of Texas Red (Taylor et al.,
1996). As these dextran-conjugated dyes leaked from the cell
wall within 10-20 minutes when the roots were washed with
the dye-free medium, experiments were conducted with the
indicator dyes still present in the medium around the root. We
therefore used the optical sectioning ability of the confocal
microscope to distinguish medium fluorescence from that of
indicator in the epidermal cell wall. Unfortunately the need for
continued dye presence in the medium precluded imaging pH
in the exposed walls of the tip growing root hair. Therefore
analysis of wall pH was limited to the initiation process.
Fig. 1 shows that taking an optical section approximately 2
µm below the surface of the epidermis resolved fluorescence
from dye in the wall from the background fluorescence of the
medium. Texas Red dextran (Fig. 1B) and NERF dextran (Fig.
1C) were similarly loaded into the apoplastic space of
trichoblasts in the root hair zone of the root and were excluded
from the cytoplasm for as long as we have observed (12 hours).
For comparison, Fig. 1E shows an equivalent region of a root
stained with fluorescein diacetate (FDA), a dye that
accumulates in the cytoplasm (Huang et al., 1986). The cell
walls in this picture appear as dark regions that fail to
accumulate the dye. Exclusion of the Texas Red and
NERF dextran conjugates from the cytoplasm, and
maintained localization to the wall, was observed in
cells from the end of the elongation zone through to
the mature region of the root (Fig. 1 and data not
shown). This localization of indicator allowed us to
monitor wall pH from just before root hair initiation
(elongation zone cells), to past the end of the initiation
process (mature region of the root).
We next ensured that infiltration of dextranconjugated dye and subsequent fluorescence imaging
did not affect root growth or root responses. The
growth rate of roots loaded with the dextranconjugated dyes and subjected to confocal imaging
(201±15 µm/hour, n=7) was similar to unloaded
controls (218±18 µm/hour, n=10). In addition, loaded
roots showed identical morphology and kinetics of the
gravitropic response (Blancaflor et al., 1998) when
compared with unloaded controls (data not shown).
The kinetics of root hair initiation and growth, and
Fig. 1. Cell wall localization of dextran-conjugated NERF and Texas Red.
trichoblast and root hair morphology were also
Roots were perfused with NERF dextran and Texas Red dextran. (A) A brightunaffected (see below). We take these as indications
field image of cells shown in B and C. Fluorescence from Texas Red dextran
that the loading of the cell walls with dextran(B) and NERF dextran (C) in the cell walls of the root hair initiation zone was
conjugated dyes and subsequent ratio imaging, was
imaged using confocal microscopy. The confocal optical section shown is
neither perturbing root growth nor altering the process
approximately 2 µm from the root surface. (D) A bright-field image of cells of
of root hair initiation.
the root hair initiation zone stained with fluorescein diacetate (FDA) (E). Note
Calibration of the Nerf/Texas Red ratio to
wall pH
We then determined that the wall located dyes were
still pH responsive. The NERF/Texas Red signal was
the cell wall staining in B and C and cytoplasmic staining and exclusion from
the cell walls in E. The bottom right panel shows a magnified view of the
regions indicated in B-E. i, initiation site; m, fluorescence from medium; tr,
trichoblast; v, vacuole. Representative views of more than 50 roots. Scale bar,
20 µm.
2928 T. N. Bibikova and others
the wall showed an similar ratio value at the same buffered
pH, including the walls at the initiation site (Fig. 2B). This
uniform in vivo calibration was observed in the walls of
trichoblasts throughout the initiation process, suggesting that
different regions of the wall were not modifying the
responsiveness of the NERF/Texas Red indicator system to
pH. Although we have attempted to calibrate the
responsiveness of the dyes in their intact wall setting, the
absolute pH values quoted should be viewed
as approximations of wall pH.
As expected, treatment with 100 nM of the
H+-ATPase activator fusicoccin led to a
reduction in wall pH of between 0.5 and 1 pH
unit (data not shown), consistent with the wall
localized NERF/Texas Red responding to
physiologically relevant changes in wall pH. In
addition, when pH buffer was applied to the
root, a change in wall pH was detectable (Fig.
2C), indicating that the ratio imaging approach
was capable of detecting changes in wall pH.
Thus the NERF/Texas Red ratio imaging
approach appeared valid for monitoring changes
in pH along the length of the cell as root hair
initiation proceeded.
Local wall acidification is associated
with root hair initiation
Figs 2D and 3 show that as trichoblasts enter the
zone of root hair initiation, the pH of the
trichoblast cell wall away from the initiation site
(middle in Fig. 3A,B) increases to approximately
pH 6 over 40-60 minutes. However, a region of
≤pH 5 is maintained at the site of root hair
initiation. The pH of this initiation region drops to
4.5 over the initial 30 minutes of root hair
initiation, which corresponds to the period of
bulging of the wall that characterizes the initiation
phase of root hair formation. When this initiation
process was stopping and the root hair was
switching from initiation to tip growth
(approximately 40-60 minutes after the wall
bulging had commenced (Fig. 3C), the locally
reduced pH at the initiation site increased to the
pH 6 found in the rest of the wall (Figs 2D, 3B).
Close inspection of the ratio images of wall
pH in Fig. 2D shows some heterogeneity in wall
pH of approximately ±0.2 pH units around the
mean pH value for any region of the wall. The
source of this variability is unknown. It does not
arise from noise due to a low signal strength
from the pH sensing dyes and is also not evident
in the images of the strongly pH buffered walls
in Fig. 2B. This variability may reflect natural
microdomains of altered apoplastic pH due to
the complex structure of the wall.
Manipulating apoplastic pH alters root
hair initiation and tip growth
To determine whether the lowered wall pH
maintained at the root hair initiation site was
involved in the initiation events or merely
coincided with them, we attempted to inhibit acidification of
this wall region by buffering the apoplastic pH. We therefore
applied the 50 mM HEPES, MES and DMGA buffers found to
effectively alter wall pH in the in situ calibration shown in Fig.
2B. The growth rate shown in Fig. 4A was calculated 30
minutes after initiation had commenced but is representative of
the effect of altering external pH at all times during the
initiation process. When apoplastic pH was buffered between
Fig. 2. Calibration of the NERF dextran/Texas Red dextran wall pH measurements.
(A) The pH dependency of the NERF/Texas Red ratio was calibrated against pH in
vitro using 1 µg/ml of each dextran, 100 mM KCl, 1 mM MgCl2, and 10 mM of
appropriate pH buffer (pH 4-5: citrate or DMGA; pH >5 MES). The in situ
calibration was obtained by infiltrating the dyes into cell walls of roots killed by
freeze thawing and incubation in ice cold ethanol. The roots were then incubated in
50 mM of pH buffer as for the in vitro calibration and mean ratio values calculated
from regions of the wall encompassing >1000 pixels using IPLabs image analysis
software. Mean ± s.e.m., n>12. Linear regression was calculated using Microsoft
Excel and least squares fitting. R2 in situ, 0.93; in vitro, 0.97. (B) Ratio images of the
in vivo calibration of the NERF/Texas Red ratio. The dyes were infiltrated into cell
walls of living roots which were then incubated in 50 mM of the appropriate pH
buffer for 15 minutes. Note the homogeneous change in wall pH throughout the wall,
including the initiation site (i). (C) The cell walls of a trichoblast were loaded with
NERF and Texas Red dextrans and pH determined by confocal pseudo-ratio imaging.
Wall pH was then altered by applying 50 mM MES, pH 6.0 and imaged after 5
minutes. Note the rapid alkalinization of the cell wall induced by this change in
apoplastic pH treatment. (D) Shows ratio images of cell walls at the indicated time
after root hair initiation. Note the decrease in pH in the cell wall at the initiation site
(i) and increase in wall pH by 55 minutes after initiation has commenced. Wall pH
has been pseudo-color coded according to the inset scale. The dark region to the side
of the initiation site in the 55 minute image is due to interference with the wall pH
imaging by the long root hair that has formed and grown out over this region of the
cell wall. The images in D are from roots grown in standard growth medium (1/2
strength Epstein’s medium, 2.3 mM MES pH 5.7) without the supplemental pH
buffers used in the calibrations shown in A-C. Representative of more than 10 roots.
Scale bar, 10 µm.
pH and root hair initiation 2929
Fig. 3. pH changes in the wall and kinetics of growth of a trichoblast
during root hair initiation. The cell walls of trichoblasts were loaded
with NERF and Texas Red dextrans and pH determined by confocal
pseudo-ratio imaging. Average wall pH measurements were
calculated from the ratio images using IPLabs image analysis
software and were taken over a 10 µm region of the wall at the
initiation site or away from the initiation site in the middle of the cell
(A). Wall pH was then followed during the root hair initiation process
(B). (C) The initiation growth kinetics expressed as the length of the
bulge in the cell wall relative to the trichoblast cell surface. Insets in C
show the typical morphology of an initiating root hair and one that
has begun tip growth. Plants were grown in standard growth medium
(1/2 strength Epstein’s medium, 2.3 mM MES pH 5.7) without
supplemental pH buffers. White bars indicate the length measured for
initiating or tip growing root hair; tr, trichoblast. Results represent
mean ± s.e.m. of more than 10 roots. Scale bar, 10 µm.
4.5 and 5.5 the average rate of the wall bulging, indicative of
the initiation process, increased as the pH was lowered until at
pH 4.0 growth was inhibited (Fig. 4A). This was probably due
to a cytotoxic action of prolonged exposure to pH 4.0. The
maximal rate of initiation in this buffered system (pH 4.5)
showed no detectable alteration in kinetics (P>0.05, t-test)
from the rate seen under the ‘normal’ growth conditions used
for Arabidopsis (1/2 strength Epstein’s medium).
When the same buffer systems were used to alter wall pH
to ≥5.5, root hair initiation was inhibited. This inhibition
occurred irrespective of the stage of initiation previously
reached by the root hair (Fig. 4A and data not shown). Thus,
Fig. 4. Effect of manipulating extracellular pH on root hair initiation
and tip growth. (A) The growth rate of initiating root hairs measured
30 minutes after initiation had commenced. (B) Tip growth rate
monitored between 90 and 120 minutes after initiation. Extracellular
pH was buffered at the indicated values using 50 mM HEPES/MES
or DMGA for at least 10 minutes before the start of each
measurement. For comparison, growth rate of root hairs under
standard growth conditions (growth medium with 2.3 mM MES, pH
5.7) is shown (䊉). The detectable tip growth rate at pH 7.5 in B
arises from slow growth of 10% of the root hairs, 90% being
completely arrested in elongation. Results represent the mean ±
s.e.m., n>8 separate roots. (C) The effect on growth of increasing the
extracellular pH from 5.0 to 7.5 on root hairs at different stages of
the initiation process. Results are representative of at least 8
individual roots. For clarity, the largest s.e.m. of the data is shown as
the inset bar.
for example, a trichoblast where the lateral cell wall had not
yet begun to bulge, but where the root hair would be predicted
to start to emerge in the next few minutes, showed no bulging
at an external buffered pH of 7.0. Initiating root hairs were
arrested at the stage of initiation they had reached when the
wall pH was elevated (Figs 4A, 5). Upon returning the
extracellular pH from 7.0 to pH 5.0 (still in the MES/HEPES
2930 T. N. Bibikova and others
(C)
Length (µm)
Measuring cytoplasmic pH in Arabidopsis
root hairs
We next sought to determine if the local changes in
wall pH in trichoblasts initiating a root hair were
mirrored by changes in cytoplasmic pH.
Developing
trichoblasts
were
therefore
microinjected with the pH sensing fluorescent dye
BCECF dextran and the pH insensitive dye
rhodamine dextran, and pH monitored throughout
the initiation and tip growth process. As found
previously, microinjected, dextran-conjugated dyes
were excluded from the vacuole and remained in
the cytoplasm for as long as we have observed (>8
hours) and did not alter the process of root hair
formation and growth (Bibikova et al., 1997;
Wymer et al., 1997). Fig. 6A shows an in vitro and
in vivo calibration of the BCECF/rhodamine ratio
versus pH and indicates that this ratiometric
analysis should accurately resolve pH in the
expected cytoplasmic range (pH 7.0-8.0). Fig. 6C,D
shows that in atrichoblasts or trichoblasts prior to
root hair emergence, cytoplasmic pH was
homogeneous at approximately 7.2. However, a
localized increase in pH to 7.7±0.2 (n=11) was seen
to be associated with the site of initiation in
trichoblasts.
This
alkalinization
formed
concurrently with the first morphological
indications of bulging in the trichoblast cell wall
(Figs 6C, 7). The alkaline pH was maintained
throughout the bulging process. Cytoplasmic pH in
atrichoblasts did not detectably change during the
time when initiation was occurring in adjacent
trichoblasts (Figs 6D, 7C). Using this confocal
ratiometric approach to monitor cytoplasmic pH,
tip growing root hairs showed no detectable,
marked pH gradient (>0.1 pH unit) either acidic or
alkaline, associated with the growing root hair apex
(Fig. 6G). Root hairs that had stopped growing
showed a similar, homogeneous cytoplasmic pH of
7.1-7.2 (Fig. 6I).
Altering cytoplasmic pH inhibits tip growth but not
root hair initiation
We next asked if the alkalinization in cytoplasmic pH was
involved in the initiation process or simply coincident with it.
We applied the weak acids propionic acid and butyric acid to
alter cytoplasmic pH and determined the effect on initiation
kinetics. Butyric and propionic acid gave identical results,
therefore only the data from butyric acid experiments is
presented. Disruption of cytoplasmic pH may also be
accomplished using weak bases such as NH4Cl (Kossegarten
et al., 1997). However, this was found to be inapplicable in
pH
treatment
buffer system) initiation growth recovered and resumed
normal root hair formation with no detectable lag (Fig. 5B,C).
This indicates that the inhibition of initiation was not due to
the buffer used, but most likely to the pH set by that buffer.
The resumption of growth of initiating root hairs was observed
when the exposure to high extracellular pH was less than 3045 minutes (Fig. 5C). Longer periods of high pH treatment led
to a failure of all initiating root hairs to resume growth. In
contrast tip growth was inhibited when extracellular pH was
increased to >7.0 and irreversibly arrested at pH ≥7.5
irrespective of the time of treatment at elevated pH (Fig. 4B
and data not shown). At pH <4.5 tip growth was
inhibited and 25% (n=54) of growing root hairs
burst. No root hairs that had ceased to elongate
before the pH 4.0 treatment were observed to burst
(n=10 separate roots).
Altering wall pH with 50 mM MES/HEPES at
pH 7.5 led to no detectable change in cytoplasmic
pH monitored by BCECF dextran/rhodamine
dextran ratio analysis (n=5, see below).
Fig. 5. Reversibility of the inhibition of root hair initiation by elevated
extracellular pH. Root hair growth was monitored as the extracellular pH was
changed from pH 5.0 to 7.5 and then returned to 5.0 using 50 mM MES/HEPES
buffer. (A) Images of a time course of initiation in root hairs maintained at pH
5.0, and (B) shows the effect of an excursion to pH 7.5 over the indicated times.
Note the inhibition of initiation at pH 7.5 and recovery on return to pH 5.0.
(C) The kinetics of the resumption of growth of a root hair as pH was changed
from 5.0 to 7.5 and back to 5.0. Note the failure to resume growth when the pH
7.5 treatment is longer than 30 minutes. Results are representative of >25 root
hairs on 8 separate roots per treatment. Bar, largest s.e.m.
pH and root hair initiation 2931
these root hair experiments as the weak bases require elevated
external pH, e.g. pH 7.0, for action. This elevated external pH
could alter initiation and tip growth kinetics due to effects on
apoplastic pH. In addition weak bases have been
shown to alter vacuolar pH in root hairs (Brauer et
al., 1997).
Butyrate at 10-15 mM inhibited tip growth but
failed to prevent root hair initiation (Fig. 8). Growth
of the main root axis was also unaltered by up to 10
mM butyrate treatment (control 218±18 µm/hour; 10
mM butyrate for 2 hours, 201±11 µm/hour, n=10).
Thus at 10 mM butyrate, the root continued to
elongate and a series of initiated root hairs
accumulated along the root but these failed to start tip
growth (Fig. 8A-C). The effects of 10 mM butyrate
were reversible for the initiation process but
irreversible in tip growing root hairs. Thus initiated
root hairs recommenced growth after perfusion of the
root with butyrate free medium for >6 hours (data not
shown). Ten mM butyrate dissipated the local
elevated cytoplasmic pH at the initiation site (Fig.
8D-F) but did not affect the locally acidic wall at the
initiation site (Fig. 8G). Ten mM butyrate caused no
statistically significant change in the cytoplasmic pH
of tip growing root hairs (apical 5 µm of control:
7.3±0.13; butyrate: 7.17±0.2 pH units, P>0.05 t-test).
At higher concentrations of butyrate (e.g. 20 mM)
root hair elongation was inhibited, and elongation
growth of the main root axis was slowed (51±30
µm/hour, n=10). However, even under these
conditions, the process of root hair initiation
proceeded with no detectable alteration in kinetics or
morphology of the initiating hair (data not shown).
also suggests that the pH responsive elements of the wall are
stable. Wall polymer structure (Carpita and Gibeaut, 1993),
enzyme activities (Fry et al., 1992; Nishitani et al., 1992),
DISCUSSION
Root hair initiation involves an epidermal cell
changing its growth habit from a diffuse longitudinal
growth in the elongation zone of the root to highly
localized lateral growth that will form the tip
growing root hair. Initiation of the root hair and its
subsequent tip growth are two independent phases of
root hair formation that exhibit differences in the
genes required and in their cell biological controls
(e.g. Ridge, 1995; Wymer et al., 1997).
Although the bulging associated with root hair
initiation is preceded by a nuclear migration (Meeks,
1985; Sato et al., 1995) and the cytoskeleton has been
shown to become reoriented after the initiation
process (Emons and Derksen, 1986), the molecular
mechanisms underlying the reorientation of growth
are unknown. We have found that the site of initiation
is associated with an alkalinization of the cytoplasm
and a local wall acidification. Preventing the local
acidification of the cell wall by raising the
extracellular pH with buffers stopped the initiation
process. Importantly, this inhibition was reversible,
suggesting the effect was not due to a cytotoxic action
of the high external pH. The rapid reversibility of the
inhibition of initiation elicited by high external pH
Fig. 6. Imaging cytoplasmic pH in trichoblasts and elongating root hairs of
Arabidopsis thaliana. (A) The in vitro (䊊) and in vivo (䊐) calibration of
BCECF/rhodamine fluorescence ratio versus pH. The in vivo measurements
were made using the protonophore nigericin to equilibrate cytoplasmic pH to
that buffered in the extracellular medium. Results represent mean ± s.e.m., n≥7
for the in vitro calibration, and measurements from individual microinjected
root hairs for in vivo calibration. (B,E) Bright-field and (C,D) ratio images of a
time course of changes in cytoplasmic pH in a trichoblast (C) and atrichoblast
(D) during root hair initiation. The trichoblast or atrichoblast was microinjected
with BCECF dextran and rhodamine dextran and cytoplasmic pH monitored by
confocal pseudo-ratio imaging. Cytoplasmic pH has been pseudo-colored
according to the inset scale. Representative of 8 individual roots. (F,H) Brightfield images of root hairs shown in G and I. (G,I) ratio images of cytoplasmic
pH in a root hair exhibiting tip growth monitored after 120 and 125 minutes of
growth (G), or a fully elongated, non growing root hair (I). Representative of
more than 9 root hairs. n, nucleus; tr, trichoblast; v, vacuole. Scale bars B and I,
10 µm; D, 15 µm.
2932 T. N. Bibikova and others
Fig. 7. Changes in cytoplasmic pH in a trichoblast and atrichoblast
during root hair initiation and tip growth. Trichoblasts and
atrichoblasts were microinjected with dextran-conjugated BCECF
and rhodamine, and cytoplasmic pH determined by confocal pseudoratio imaging. In an initiating root hair cytoplasmic pH
measurements were taken at the initiation site or away from the
initiation site in the middle of the cell. For tip growing root hairs, the
pH measurements were made from the flank or tip, as indicated in
(A). Average cytoplasmic pH in trichoblasts (B) and atrichoblasts (C)
was calculated from the ratio images using IPLabs image analysis
software using a 5-10 µm region of the cytoplasm as indicated in the
insets in A. For the atrichoblast, pH at the initiation site represents
the pH in the cytoplasm at the point where a root hair would be
expected to emerge if the cell were a trichoblast. For tip growth, the
measured region at the flank of the root hair was representative of the
pH throughout the cytoplasm to the apical 10 µm region covered by
the measurement of tip pH. tr, trichoblast. Results are mean ± s.e.m.
(n=7, atrichoblasts; n=11, trichoblasts).
effects on the levels of other ions e.g. Ca2+ (Arif and Newman,
1993) and channel activities in the plasma membrane (Hedrich
and Dietrich, 1996) are all potential sites of action of altered
apoplastic pH. Additionally, acid-induced growth has been
linked to wall extensibility in roots (e.g. Pritchard, 1994), and
proteins with acidic pH optima (expansins; McQueen-Mason et
al., 1992) that can mediate wall relaxation of root cell walls
Fig. 8. The effect of 10 mM butyrate on root hair initiation and tip
growth. (A) The root hair zone of a control root and (B) one
treated with 10 mM butyric acid for 4 hours. Note the profusion of
elongated (tip growing) root hairs of the control (✩) and the
accumulation of initiated root hairs that have failed to start tip
growth (arrows) in the butyrate treated root. The elongated root
hair in the butyrate treated root (✩) was already tip growing when
the butyric acid was applied. Scale bar represents 50 µm. (C) The
effect of butyrate on the kinetics of root hair initiation and tip
growth. Roots were treated with (䊐) or without (䊏) 10 mM
butyric acid and the growth of the initiation point measured.
Results are representative of more than 8 root hairs. Bar, largest
s.e.m. (D) Bright-field image of E and F. (E,F) Ratio imaging of
cytoplasmic pH in a trichoblast before (E) and after (F) treatment
with 10 mM butyrate for 10 minutes. Note dissipation of elevated
pH at the initiation site (i) in the butyrate treated root.
Representative of 8 separate root hairs. (G) Wall pH monitored by
NERF/Texas Red ratio imaging in a trichoblast treated with 10
mM butyrate for 10 minutes and then imaged at −5 and 20 minutes
after the initiation process had commenced. Note the continued
acidification of the wall at the initiation site (i) despite butyrate
treatment. Cytoplasmic and wall pH have been color coded
according to the inset scales. Representative of more than 8
individual roots.
pH and root hair initiation 2933
have been isolated (Wu et al., 1996). Expansins have been
cloned from Arabidopsis (Scherban et al., 1995) and would be
predicted to show minimal wall relaxing activities at pH ≥7.0
(McQueen-Mason et al., 1992).
Localized cellular growth phenomena in Fucus and Pelvetia
zygotes (Kropf and Quatrano, 1987, Kropf et al., 1995),
Micrasterias (Holzinger et al., 1995) and Funaria (Demkiv et
al., 1994) have also been linked to alterations in cytoplasmic
pH. Interestingly, rhizobial nod factors alter root hair growth
habit and have been shown to elicit an elevation in cytoplasmic
pH in alfalfa root hairs (Felle et al., 1996). This elevation is
coincident with the inhibition of apical growth and the
induction of root hair deformation and curving that occurs
during nodulation. These observations suggest that the
increased cytoplasmic pH may play a role in these alterations
of root hair growth form. In contrast, we have found that the
cytoplasmic alkalinization at the root hair initiation site is not
an absolute requirement for the initiation process to proceed.
Thus blocking the increase in cytoplasmic pH with butyrate did
not prevent initiation. Similarly, raising wall pH blocked
initiation without affecting the locally elevated cytoplasmic pH
at the initiation site.
The role of cytoplasmic pH in tip growth is also unclear. The
pH-sensitive vibrating microelectrode has detected both influx
(Jones et al., 1995) and efflux (Hermann and Felle, 1995) of
protons at the growing tips of root hairs. In root hairs of Sinapis
Alba impaled with pH selective microelectrodes no pH
gradients were detected associated with tip growth (Hermann
and Felle, 1995). We were also unable to detect a pH gradient
associated with the growing tip of root hairs of Arabidopsis.
Although we cannot exclude the possibility of changes below
the spatial (<1 µm) or temporal (<1 second) resolution of our
imaging equipment were not recorded. Assuming the lack of a
pH gradient accurately reflects pH dynamics in the growing
hair, the inhibition of tip growth caused by alteration of
apoplastic pH or treatment with butyrate may well reflect
disruption of the pH homeostasis required for the tip growth
machinery to operate, rather than effects on a steady state pH
gradient at the tip. A similar conclusion was drawn by
Hermann and Felle (1995) for the effects of manipulating
cytoplasmic pH on tip growth in root hairs of Sinapis Alba. In
addition, we noted that at pH <4.5, growing root hairs often
burst at the tip whereas root hairs that had previously stopped
growing were unaffected. Lowering the extracellular pH to
below 4.5 may weaken the pH dependent events regulating
structure of the wall at the tip of the hair to the point where
turgor causes bursting. The wall polymers at the tip of the non
growing root hairs might then be modified to be insensitive to
this pH change as a normal part of the mechanism whereby
growth is arrested.
Whether the localized pH changes at the initiation site
reflect localized changes in wall polymer structure and ion
exchange capacities (Cosgrove, 1997) or localized activation
of the H+-ATPase or other H+ transporting activity is
unknown. We have found agents that should alter H+-ATPase
activity, such as vanadate and cyanide do inhibit the initiation
process, but also rapidly (<30 minutes) inhibit root growth
(data not shown). Such manipulations may be altering root
hair initiation by disrupting other root processes rather than a
direct action on the H+-ATPase at the initiation site. Therefore
they do not provide reliable evidence for a role of the H+-
ATPase in this process. However, a plasma membrane H+ATPase has been immunolocalized in barley root hairs
(Samuels et al., 1992) and root hairs show a detectable H+
current at their apex (Weisenseel et al., 1979; Jones et al.,
1995). In addition, a H+-ATPase is localized to the apical
plasma membrane of another tip growing system, pollen tubes
(Obermeyer et al., 1992). Putative mechanisms for a localized
activation of the H+-ATPase at the initiation site include
reversible inhibition by submicromolar Ca2+ (Kinoshita et al.,
1995) or Ca2+-dependent phosphorylation (Schaller and
Sussman, 1988). Thus although our data suggest the potential
activity of the H+-ATPase in the root hair initiation process,
further studies are needed to precisely define its role.
Our results indicate apoplastic pH is intimately linked with
localized growth of the initiating root hair. However, the root
hair initiation process is a complex reorientation of growth that
is likely to involve many interacting regulatory systems and
localized wall modifications. Thus we found that simply
reducing wall pH with a pH buffer did not trigger or accelerate
the initiation process, nor did it lead to inappropriate bulging of
the trichoblast wall. The nuclear migration that precedes
initiation (Meeks, 1985; Sato et al., 1995) suggests the events
determining the initiation site are laid down long before the first
morphological indications of wall bulging. Consistent with this
idea, we have been unable to detect a distinct pH change in
either the wall or cytoplasm predicting the initiation site.
However, although localized pH changes in the wall and
cytoplasm do not appear to determine the initiation site, our
results strongly suggest the pH changes in the wall are
intimately involved in the machinery that translates the signals
specifying the initiation site into the process of root hair
emergence. In contrast, although we could not detect localized
pH gradients associated with tip growth in root hairs, treatments
that should disrupt apoplastic and cytoplasmic pH dynamics
both inhibit the tip growth process. These results further support
the genetic data (Ridge, 1995) that tip growth and root hair
initiation are very different processes and suggest that control
of both cell wall and cytoplasmic pH dynamics is essential in
maintaining tip growth.
The authors would like to thank Sian Ritchie and Elison Blancaflor
for critical reading of the manuscript. This work was supported by
grants from the Studienstiftung des Deutschen Volkes (TJ), the
USDA/DOE/NSF Interdisciplinary Research Training Group in
Advanced Root Biology (BIR-9220330; T. N. B.), and NSF (IBN9513991; S. G.).
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