Download Get PDF file - Botanik in Bonn

Planta (1999) 209: 435±443
Redistribution of actin, pro®lin and phosphatidylinositol-4,5-bisphosphate in growing and maturing root hairs
Markus Braun, Frantisek BalusÏ ka, Matthias von Witsch, Diedrik Menzel
Botanisches Institut, Zellbiologie der P¯anzen, UniversitaÈt Bonn, Venusbergweg 22, D-53115 Bonn, Germany
Received: 11 March 1999 / Accepted: 27 May 1999
Abstract. The continuously changing polar cytoplasmic
organization during initiation and tip growth of root
hairs is re¯ected by a dynamic redistribution of cytoskeletal elements. The small G-actin binding protein,
pro®lin, which is known to be a widely expressed, potent
regulator of actin dynamics, was speci®cally localized at
the tip of root hairs and co-distributed with a di€usely
¯uorescing apical cap of actin, but not with subapical
actin micro®lament (MF) bundles. Pro®lin and actin
caps were present exclusively in the bulge of outgrowing
root hairs and at the apex of elongating root hairs; both
disappeared when tip growth terminated, indicating a
tip-growth mechanism that involves pro®lin-actin interactions for the delivery and localized exocytosis of
secretory vesicles. Phosphatidylinositol-4,5-bisphosphate (PIP2), a ligand of pro®lin, was localized almost
exclusively in the bulge and, subsequently, formed a
weak tip-to-base gradient in the elongating root hairs.
When tip growth was eliminated by the MF-disrupting
inhibitor cytochalasin D, the apical pro®lin and the actin
¯uorescence were lost. Mastoparan, which is known to
a€ect the PIP2 cycle, probably by stimulating phospholipases, caused the formation of a meshwork of distinct
actin MFs replacing the di€use apical actin cap and,
concomittantly, tip growth stopped. This suggests that
mastoparan interferes with the PIP2-regulated pro®linactin interactions and hence disturbs conditions indispensable for the maintenance of tip growth in root hairs.
Key words: Actin ± Cytochalasin D ± Mastoparan ±
Phosphatidylinositol-4,5-bisphosphate ± Pro®lin ± Tip
growth
Abbreviations: FITC = ¯uorescein isothiocyanate; MF = micro®lament; PIP2 = phosphatidylinositol-4,5-bisphosphate
Correspondence to: M. Braun
E-mail: [email protected]; Fax: 49 (228) 732677
Introduction
The cytoplasmic architecture of plant cells is highly
dynamic and depends on continuous remodelling of
cytoskeletal elements in response to developmental and
environmental signals and requirements. The arrangement and properties of the cytoskeleton are known to be
regulated by associated proteins that bind either to
monomeric or polymerized cytoskeletal proteins. The
number of well studied and characterized cytoskeletonregulating proteins in plant cells is still very limited (for
review, see Staiger et al. 1997; De Ruijter and Emons
1999). Pro®lin, a widely expressed G-actin-binding
protein, which interacts with poly-L-proline and
phosphoinositides (Sun et al. 1995; SchluÈter et al.
1997; Chaudhary et al. 1998; Gibbon et al. 1998) is
one of the best-characterized examples. Isoforms of
pro®lin have been reported to regulate cellular growth
and morphogenesis by organizing actin cytoskeletal
dynamics in higher plants (e.g. Staiger et al. 1994,
1997; Valster et al. 1997). The actin-binding properties
of plant pro®lins have been demonstrated in vitro
(Valenta et al. 1993; Giehl et al. 1994; Staiger et al.
1994; Vidali and Hepler 1997) and in situ by microinjection experiments (Cao et al. 1992; Staiger et al. 1994;
Gibbon et al. 1997, 1998; Holzinger et al. 1997; Valster
et al. 1997). The distribution of pro®lin in plant cells,
however, has not been studied in great detail so far.
Previous localization studies in pollen tubes (Mittermann et al. 1995; Vidali and Hepler 1997) and the green
alga Micrasterias (Holzinger et al. 1997) have shown
that pro®lin is evenly distributed throughout the cytoplasm and is not associated with any cellular structure.
Nevertheless, a possible role of pro®lin as part of the
signalling pathways involved in the regulation of pollen
tube tip growth has been suggested (Clarke et al. 1998a).
On the other hand, in animal cells such as ®broblasts,
the distribution of pro®lin correlates with highly dynamic F-actin arrays but not with relatively stable actin
micro®laments (MFs), suggesting that pro®lin is an
essential partner involved in the regulation of actin
dynamics (Buss et al. 1992).
436
Root hair initiation is characterized by the shift of the
cellular polarity from di€use expansion growth of the
trichoblasts to localized and highly polarized tip growth.
On the light-microscopical level, the bulging of the cell
at the site of root hair emergence is the ®rst visible event.
However, a number of intracellular events precede the
actual outgrowth of the root hair, e.g. the sudden onset
of cytoplasmic streaming, reorientation of cytoplasmic
strands and endoplasmic reticulum, and a major rearrangement of cytoskeletal elements and associated
proteins. During the various developmental stages of
tip growth in root hairs (see Fig. 1 in Heidstra et al.
1997), the polar organization of the di€erent cytoplasmic zones, including the apical vesicle-rich region, as
well as the pattern of cytoplasmic streaming and the
arrangement of the actin cytoskeleton change constantly
(Miller et al. 1997, 1999).
In this study, root hairs of three plant species were
used for investigating the dynamic redistribution of
actin, pro®lin and phosphatidylinositol-4,5-bisphosphate (PIP2) and the possible involvement of pro®lin
in the regulation of actin-dependent processes in tipgrowing plant cells. Cytochalasin D was used to interfere
with the actin cytoskeleton and mastoparan to interfer
with the possible PIP2-regulated pro®lin-actin interaction. Our experiments, which include phalloidin staining
and immunolabeling of actin, pro®lin and PIP2 in
untreated and inhibitor-treated root hairs in di€erent
developmental stages of root hairs, indicate that pro®linactin interactions have an important function in tip
growth of root hairs.
Materials and methods
Maize (Zea mays L. cv. Alarik) grains were obtained from Force
Limagrain (Darmstadt, Germany), Lepidium sativum L. (cress)
seeds from Chrysanth, Bonn, Germany and Arabidopsis thaliana
(L.) Heynh. seeds from Lehle Seeds, Round Rock, USA. Maize
grains and cress seeds were soaked for 6 h and germinated in
moistened rolls of ®lter paper for 2±3 d in darkness at 20 °C.
Arabidopsis seeds were germinated on agar plates containing 1%
sucrose. For inhibitor treatment, germinated seedlings were adapted
to aerated water for 6 h and then incubated with cytochalasin D (5±
10 lM; Sigma-Aldrich Chemie, Deisenhofen, Germany) and mastoparan (1±5 lM; Sigma) for 30 min prior to ®xation. For in-vivo
observations, seedlings were mounted on a slide in a drop of water,
covered with a coverslip, and inspected under a microscope
connected to a video recorder. Inhibitors were applied by adding
them at working concentration directly to the mounted roots.
Fig. 1A±G. Detection of a pro®lin band at approx. 14 kDa by the
anti-pro®lin (ZmPRO3) antibody in root extracts of Arabidopsis (A),
maize (C soluble proteins, E microsomal proteins) and cress (G).
Preincubation of anti-ZmPRO3 with recombinant pro®lin isoforms
resulted in strongly reduced labeling of the pro®lin band of the soluble
protein fraction of maize (D) and a complete suppression of the
labeling in the maize microsomal fraction (F) and in the Arabidopsis
(B) total protein fraction
M. Braun et al.: Actin, pro®lin and PIP2 in root hairs
Freeze-shattering and confocal microscopy. The freeze-shattering
procedure was modi®ed after Braun and Wasteneys (1998). Either
whole roots (Arabidopsis) or epidermal strips with subjacent cortical
cell layers excised from roots (Lepidium, Zea), untreated and
inhibitor-treated, were ®xed for 30 min with a freshly prepared
®xation solution containing 1% formaldehyde and 1% glutaraldehyde (Sigma; Grade I, stored at )20 °C), 50 mM Pipes, 5 mM
EGTA and 5 mM MgSO4 (pH 7.2). After several rinses in ®xation
bu€er without aldehydes, the bu€er was gradually replaced with
phosphate-bu€ered saline (PBS: 137 mM NaCl, 2.7 mM KCl,
4.9 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4), incubated three
times with freshly prepared 1 mg/ml NaBH4, washed again in PBS,
placed into cold ()10 °C) methanol for 5 min and rinsed with PBS
containing 50 mM glycine. After permeabilization with 1% Triton
X-100 in PBS/glycine for 30 min, the roots were gently squashed
between two polyethyleneimine-coated microscope slides and
plunged into liquid nitrogen for approx. 1 min. The frozen slides
were rapidly separated, but then joined back together again and
pressed in order to fracture the frozen tissue. After thawing, the root
fragments were incubated with the ®rst antibodies, anti-actin (clone
C4 from ICN, Costa Mesa, USA; 1:400), anti-pro®lin (maize
isoform ZmPRO3; Karakesisoglou et al. 1996; 1:200), anti-PIP2
(Perseptive Biosystems, Framingham, USA; Bubb et al. 1998;
1:200) for 3 h at 37 °C or overnight at room temperature. After
three rinses in the same bu€er, the cells were incubated with
¯uorescein isothiocyanate (FITC)-conjugated second antibodies for
2 h (Sigma, 1:100) at 37 °C. For double-labeling of pro®lin and
actin, root fragments were sequentially incubated with anti-pro®lin
for 10 h, with anti-actin (C4) for 3 h, with FITC-conjugated antirabbit (Sigma) for 2 h and with Alexa 546-conjugated anti-mouse
(Molecular Probes, Eugene, Ore., USA) for 2 h. Washing was
performed after each incubation step. Stained samples were rinsed
three times with PBS/glycine and mounted in 0.1% para-phenylene
diamine and 50% glycerol to minimize fading of the ¯uorescent
conjugate. Images of immuno¯uorescently labeled samples were
collected using a confocal microscope (TCS4D; Leica, Heidelberg,
Germany). Several controls were performed to determine the
speci®city of the antibody labelings, including incubation with
bu€er only and with primary antibodies only as well as incubation
with secondary antibodies only. Preabsorption of anti-ZmPRO3
with recombinant pro®lins was carried out by incubating 1 ml of
diluted antibody with 60 lg of ZmPRO3 and 20 lg of ZmPRO1, 2,
and 4 overnight at 4 °C. Anti-PIP2 (Perseptive Biosystems) was
preabsorbed with PIP2 from bovine brain (Sigma) overnight at
4 °C. Recombinant Pro®lin was also used as an inappropriate
antigen to preabsorb the anti-PIP2-antibody and PIP2 was used as
an inappropriate antigen to preabsorb anti-ZmPRO3.
Oregon-green phalloidin and rhodamine phalloidin (both Molecular Probes) stock solutions (0.3 lM, in 100% methanol) were
diluted 1:100 with SoÈrensen-phosphate bu€er (pH 7.2) and
labelings were imaged using the confocal microscope.
Preparation of protein extracts and immunoblotting. Roots of 3-dold maize and cress seedlings grown on wet ®lter paper and whole
seedlings of Arabidopsis thaliana grown on agar for 5 d were
collected and homogenized in a bu€er containing 50 mM Tris (pH
7.4), 300 mM sucrose, 5 mM KCl, NaCl and EDTA, 2 mM
ascorbic acid, 10 mM freshly added DTT, and a cocktail of
protease inhibitors (10 lg/ml each of pepstatin A, leupeptin,
aprotinin, benzamidin and 1 lg/ml phenanthrolin). The Arabidopsis
and cress homogenates were each centrifuged for 10 min at 20,000 g
and 4 °C to remove cell debris and large organelles. The resulting
supernatants, containing membrane vesicles and soluble proteins,
were subjected to SDS-PAGE, using 15% mini slab gels at 15 lg of
protein per lane. Gels were wet-blotted onto nitrocellulose, which
was used for incubation with anti-ZmPRO3 and preimmune serum
at a dilution of 1:500 in TTBS (15 mM Tris, 150 mM NaCl, 0.05%
Tween 20, pH 7.4). As a control, preabsorption of anti-ZmPRO3
with recombinant pro®lins was carried out by incubating 1 ml of
diluted antibody with 60 lg of ZmPRO3 and 20 lg of ZmPRO1, 2,
and 4. The maize homogenate was centrifuged at 6,000 g for 15 min
M. Braun et al.: Actin, pro®lin and PIP2 in root hairs
at 4 °C to remove cell debris and large organelles. The pellet was
discarded and the supernatant was then centrifuged at 100,000 g for
60 min at 4 °C. This supernatant containing soluble proteins and
the pellet containing microsomal proteins were subjected to SDSPAGE and immunoblotting as described above.
Results
Immunoblot analysis. The antibody against the pro®lin
isoform ZmPRO3 (Karakesisoglou et al. 1996) recognized a polypeptide band at approx. 14.2 kDa in root
extracts of all three species, Arabidopsis (Fig. 1A), maize
(Fig. 1C,E) and cress (Fig. 1G). For a control, antiZmPRO3 antibody solution was preincubated with the
recombinant maize pro®lin isoforms ZmPRO1, 2, 3
and 4. This resulted in a strongly reduced pro®lin
labeling in the fraction of soluble maize proteins
(Fig. 1D) and even a complete inhibition of labeling in
the fraction of microsomal maize proteins (Fig. 1F) and
Arabidopsis proteins (Fig. 1B). Pro®lin labeling in the
soluble protein fraction (Fig. 1C) and the microsomal
fraction (Fig. 1E) indicates that pro®lins are not only
associated with the plasma membrane but that they are
also present in the cytoplasm.
437
Root hair initiation. The results obtained by immuno¯uorescence labeling were mostly identical in the three
species, cress, maize, and Arabidopsis and are, therefore,
not discussed in detail for each of the species, unless
necessary. A dramatic reorientation of the actin cytoskeleton was one of the ®rst characteristic features of the
initiation of root hairs following the appearance of a
bulge in root epidermal cells. After the appearance of the
bulge, the mainly longitudinally arranged actin MF
bundles of the epidermal cell surrounding the large
vacuole and the nucleus (Fig. 2) begin to focus towards
the bulge of the outgrowing root hair (Figs. 3A, 4). The
apex of the immuno¯uorescently labeled outgrowing
root hair itself did not contain distinct actin MFs, but a
brightly ¯uorescing cap of di€usely labeled actin
(Fig. 3A). Double-labeling with anti-actin and antipro®lin (ZmPRO3) antibodies revealed a cap-like profilin pattern (Fig. 3B) which spatially co-localized with
the di€usely ¯uorescing actin cap (Fig. 3A), but not with
the actin MF bundles. Neither of the cap-like ¯uorescence patterns coincided completely with the apical
cytoplasmic portion; they constituted only a small, distal
part of the apical cytoplasm. An actin depletion of the
vesicle-rich region at the outermost tip as was reported
by Miller et al. (1999) but was not observed here.
Fig. 2. Immuno¯uorescence image of the actin distribution in cress
epidermal root cells. The mainly longitudinally oriented actin MFs
reverse direction at the cross-walls
Fig. 3A±C. Double-labeling of actin (A) and pro®lin (B) in cress
root trichoblasts during root hair initiation. The corresponding
bright®eld image is shown in C. Actin MFs run into the bulge of the
outgrowing root hair and merge in a di€usely ¯uorescing cap of actin
at the tip (A) which co-localizes with the cap-like pro®lin
immuno¯uorescence (B)
Fig. 4. Immuno¯uorescence image showing the actin redistribution
in maize epidermal cells during the outgrowth of root hairs from
bulges
Fig. 5. Immuno¯uorescence labeling of PIP2 in a maize trichoblast.
The nucleus (N ), the outgrowing root hair and the cytoplasmic
portion beneath show bright ¯uorescence signals
Figs. 2±5. Bars = 10 lm
438
Phosphatidylinositol-4,5-bisphosphate (PIP2) epitopes
were immuno¯uorescently localized exclusively in the
outgrowing bulge, in the cytoplasmic portion beneath
the bulge and in the nucleus (Fig. 5). Optical sectioning
by confocal microscopy revealed that PIP2 was not
exclusively associated with the plasma membrane but is
also present in the cytoplasm.
Control experiments in which samples were incubated
with bu€er only, with primary antibodies only, and with
secondary antibodies only, as well as incubating samples
with the preabsorbed antibodies followed by the secondary antibody, resulted in no or little background
¯uorescence (not shown). Using inappropriate antigens
to preabsorb the anti-PIP2 and anti-ZmPRO3 antibody
did not a€ect the speci®c staining (not shown). These
control experiments demonstrate the speci®city of the
antibodies used in this study. In addition, the anti-PIP2
antibody has previously been shown to bind exclusively
to PIP2 (Bubb et al. 1998).
Elongating root hairs. In elongating cress root hairs, the
immuno¯uorescently labeled di€use actin cap is more
prominent and appears to be separated from the axially
oriented bundles of actin MFs by a gap zone with
strongly reduced or even absent actin immuno¯uorescence (Fig. 6A). Although such a gap was found in fastgrowing root hairs of all three species, it was less clearly
recognizable in root hairs of maize and Arabidopsis.
Labeling of the actin cytoskeleton by Oregon-green
phalloidin and rhodamine phalloidin (Fig. 7) was similar to the immuno¯uorescence labeling (Fig. 6A), but
the apical actin cap had a more ®lamentous appearance
and no gap beneath the actin cap was visualized. Median
optical sections demonstrate that actin is spread
throughout the whole distal portion of the apical
cytoplasm (Fig. 6A), whereas pro®lin ¯uorescence is
most prominent near the apical membrane (Figs. 6B,
8B). The pro®lin caps were more pronounced in cress
(Fig. 6B) and Arabidopsis (Fig. 8A) root hair tips than
in those of maize (Fig. 8B), where pro®lin immuno¯uorescence was restricted to the outermost apical plasma
membrane.
In elongating root hairs, anti-PIP2 immuno¯uorescence was found distributed uniformly throughout the
cytoplasm of short root hairs (Fig. 9A, arrow). In most
longer root hairs (Fig. 9A, arrowhead), the immuno¯uorescence was brightest at the apex. In fully grown,
mature root hairs, PIP2 was no longer observed
(Fig. 9B).
Tip-growth-terminating root hairs. Following the rapidelongation growth phase, root hairs showed reduced
growth rates and eventually tip growth stopped. In root
hairs that were terminating tip growth, the actin-depleted
subapical zone disappeared and the longitudinal actin
MFs made contact with the actin cap which became
smaller and ®nally disappeared (Fig. 10A). Simultaneously, the cap-shaped anti-pro®lin ¯uorescence was
strongly reduced (Fig. 10B) and disappeared after tipgrowth had stopped. The nucleus, which had become
spindle-shaped after entering the root hair, still showed
M. Braun et al.: Actin, pro®lin and PIP2 in root hairs
c
Fig. 6A,B. Double-labeling of actin (A) and pro®lin (B) in short,
rapidly elongating cress root hairs. A Actin immunolabeling after
freeze-shattering reveals a clear zonation of the cytoarchitecture. Actin
MF bundles are present in the trichoblast and in the base of the root
hair. An actin-depleted area in the subapical region separates the actin
MF bundles from the bright, di€usely ¯uorescing cap of actin in the
tip. Projection of 12 serial sections (1.0 lm each). B Pro®lin
immuno¯uorescence is highest near the apical plasma membrane,
but is also present in the apical cytoplasm. Projection of 3 median
serial sections (0.8 lm each)
Fig. 7. The Oregon-green phalloidin labeling pattern of short maize
root hairs resembles that of the immunolabeling except for the
absence of a subapical actin-depleted zone
Fig. 8A,B. Immunolocalization of pro®lin in root hairs of Arabidopsis (A) and maize (B) after freeze-shattering. A The cap-like pro®lin
immuno¯uorescence is most extensive in the apex of rapidly
elongating root hairs (arrows); ¯uorescence is reduced in long,
growth-terminating root hairs (arrowheads) and hardly visible in root
hairs which have terminated tip growth and have lost their polar
cytoplasmic zonation (asterisks). Projection of 10 serial sections
(1.2 lm each). B Pro®lin immuno¯uorescence in maize root hairs is
limited to a narrow region at the apical cytoplasmic membrane.
Projection of 4 median serial images (0.8 lm each)
Fig. 9A,B. Immuno¯uorescence image showing the distribution of
PIP2 in growing (A) and in mature, non-growing (B) maize root hairs.
A In short root hairs, PIP2 is evenly distributed but forms a weak tipto-base gradient in longer maize root hairs. Projection of 6 serial
images collected at 0.8-lm intervals. B Mature root hairs do not show
a speci®c ¯uorescence pattern. Projection of 6 serial images collected
at 0.8-lm intervals
Fig. 10A,B. Immuno¯uorescence labeling of actin (A) and pro®lin
(B) in a growth-terminating Arabidopsis root hair. A Actin MF
bundles have invaded the apical dome. B Pro®lin labeling is limited to
the spindle-shaped nucleus and a strongly reduced, weakly ¯uorescing
apical cap. Both images are projections of 10 serial sections (0.8 lm
each)
Fig. 11. Thick bundles of actin MFs reverse at the tip of a full-grown
cress root hair and cytoplasmic streaming occurs up to the outermost
tip. Projection of 8 serial sections (0.8 lm each)
Fig. 12. Pro®lin immuno¯uorescence is no longer visible in fullgrown cress root hairs. Projection of 8 serial sections (0.8 lm each)
Figs. 6±12. Bars = 10 lm
anti-pro®lin ¯uorescence (Fig. 10B). In mature, nongrowing root hairs, thick longitudinally oriented bundles
of actin MFs continued into the apex, performed U-turns
and returned back to the base of the cell (Fig. 11). Pro®lin
(Fig. 12) and PIP2 immunolabeling (Fig. 9B) could no
longer be detected in the tips of mature root hairs.
Inhibitor studies. Application of cytochalasin D (5±
10 lM) stopped tip growth of root hairs and resulted in
a complete depolymerization of the actin MFs in root
epidermal cells (Fig. 13A,B) as well as in root hairs
(Fig. 14A). The cells were depleted of actin immuno¯uorescence except for thick spike-like structures of actin
within the nucleus of root epidermal cells (Fig. 13A,B).
Cytochalasin D also a€ected the distribution of pro®lin.
The cap-shaped pattern was gradually replaced by a
punctate pattern in the subapical region (Fig. 14B)
before it ®nally disappeared completely (not shown).
Treatment with mastoparan, which was applied for
30 min prior to ®xation, resulted in fast termination of
M. Braun et al.: Actin, pro®lin and PIP2 in root hairs
tip growth accompanied by a drastic rearrangement of
the actin cytoskeleton in the apex. Instead of the di€use
actin cap, a distinct actin MF network was visualized
after mastoparan treatment in the bulge of outgrowing
(Fig. 15, arrows) and the tip of longer root hairs
(Figs. 16A, 17). The pro®lin cap dispersed and ®nally
disappeared completely (Fig. 16B). The cytoplasmic
streaming, however, was not visibly a€ected by mastoparan treatment.
Discussion
The polarity and cytoplasmic architecture of di€usely
expanding root epidermal cells changes dramatically
439
with the appearance of a bulge at the site of root hair
outgrowth. The emergence of the tube-like root hair
requires assembly of a tip-growth machinery for localized exocytosis of cell wall and membrane material.
Actin MFs have been localized in root hairs and appear
to be essentially involved in the cytoplasmic organization and the process of tip growth, despite the fact that
actin MFs have never been shown to extend up to the
extreme tips of elongating root hairs (Seagull and Heath
1979; Emons 1987; Ridge 1988), whereas this feature is
typical of mature root hairs (CaÂrdenas et al. 1998). It
has been shown that tip growth of root hairs, but not the
formation of bulges from which the root hairs originate
require a tip-focused gradient of calcium (Wymer et al.
1997; De Ruijter et al. 1998) and an apical array of ®ner
440
Fig. 13A,B. Cytochalasin D treatment causes complete disruption of
actin MFs in maize epidermal cells (outlined). Instead, spike-like actin
structures are formed in mitotic (A) and interphase nuclei (B).
Projection of 8 serial images (1.0 lm each)
Fig. 14A,B. Double-labeling showing the distribution of actin (A)
and pro®lin (B) in cytochalasin D-treated cress root hairs. A Actin
MFs and the apical actin cap of cress root hairs disappeared
completely after treatment with cytochalasin D. B The apical cap-like
pro®lin immuno¯uorescence is reduced and a punctate staining
pattern appeared in the subapical zone. Projection of 10 serial images
(0.8 lm each)
Fig. 15. Actin-immunolabeling of maize epidermal cells with bulges
of outgrowing root hairs (arrows) after treatment with mastoparan.
The apices contain a meshwork of actin MF bundles instead of the
di€use cap-like actin ¯uorescence in untreated root hairs (Figs. 3A, 4).
Projection of 6 serial images (1.2 lm each)
Fig. 16A,B. Double-labeling of actin (A) and pro®lin (B) in
mastoparan-treated cress root hairs. A The apical actin cap has
disappeared, but the actin MF bundles are still present. B A speci®c
pro®lin distribution is no longer visualized in the mastoparan-treated
root hair. Projections of 10 serial images (1.2 lm each)
Fig. 17. The maize root hair had stopped tip growth after mastoparan treatment for 30 min and was subsequently immuno¯uorescently
labeled for actin. Thick actin MF bundles have split into a dense
apical meshwork of ®ner actin MFs which have replaced the former
di€use actin cap in the apex. Projection of 10 serial images (0.8 lm
each)
Figs. 13±17. Bars = 10 lm
M. Braun et al.: Actin, pro®lin and PIP2 in root hairs
bundles, including an actin-depleted area at the outermost tip (Miller et al. 1999).
In this study, we have demonstrated a drastic
rearrangement of the actin cytoskeleton following bulge
formation, and the presence of di€usely ¯uorescing actin
caps in the bulges of outgrowing root hairs. Actin MFs
become focused towards the site of outgrowth which is
preceded and accompanied by the onset of vigorous
cytoplasmic streaming in the trichoblasts. Di€use apical
actin ¯uorescence was detected not only during the early
developmental stages, but also in elongating and maturing root hairs. Intriguingly, in tip-growth-terminating
and non-growing root hairs, devoid of the apical
cytoplasmic and vesicle-rich region, actin caps were
not observed. Instead, actin MF bundles formed in the
apical dome, as was also reported by Miller et al. (1999).
In elongating root hairs the patterns of apical actin
visualized by immuno¯uorescence and phalloidin labeling suggest that the actin cap is composed of both
monomeric actin and delicate actin MFs. An actindepleted zone spatially coinciding with the vesicle-rich
region was not observed with our freeze-shattering
method which avoided cell wall digestion with enzymes,
but included chemical ®xation.
Immuno¯uorescence double-labeling demonstrates
that pro®lin co-localizes exactly with the di€usely
M. Braun et al.: Actin, pro®lin and PIP2 in root hairs
¯uorescing actin cap in the tip of outgrowing and
elongating root hairs. This pattern was most easily
observed in cress root hairs, whereas it was less
prominent in maize and Arabidopsis. This speci®c apical
co-localization of actin and pro®lin is strictly correlated
with the process of tip growth. With the outgrowth of
root hairs both the actin and the pro®lin caps appeared
after bulge formation and became progressively reduced
as root hairs approached termination of tip growth.
It has been argued that pro®lin can have at least dual
e€ects: actin sequestration and actin polymerization. On
the one hand, pro®lins can bind G-actin, forming 1:1
complexes, thereby initiating disassembly or preventing
actin MF assembly (Cao et al. 1992; Fechheimer and
Zigmond 1993; Valenta et al. 1993). On the other hand,
however, pro®lin can stimulate and promote actin
polymerization at the barbed end (Tilney et al. 1983;
Pollard and Cooper 1984; Pring et al. 1992; Perelroizen
et al. 1996) depending on the cellular location and the
presence of other actin-binding proteins (reviewed by
Staiger et al. 1997). Thus, in root hairs, the high
concentration of pro®lin-actin complexes in the apex
could contribute to actin MF elongation towards the
subapical part of root hairs. Pro®lin could also mediate
cytoskeleton-membrane interactions when associated
with the plasma membrane by binding actin and
phosphoinositides simultaneously (Hartwig et al. 1989;
Machesky and Pollard 1993), thereby coordinating fast
delivery and apical exocytosis of secretory vesicles. Such
a scenario corresponds well with the observation that
pro®lin and actin caps are most prominent in fastgrowing root hairs, whereas both become smaller and
disappear when tip growth slows down and ®nally stops.
In these cells, actin MFs form thick undulating bundles
in the apex which U-turn in the outermost tip; the
rotational cytoplasmic streaming runs through the
outermost tip and polar cytoplasmic organization is lost
(see Fig. 1 in Heidstra et al. 1997).
Phosphatidylinositol-4,5-bisphosphate, which is discussed as a regulator involved in pro®lin-actin interactions (De Corte et al. 1987; Hansson et al. 1988; Drobak
and Watkins 1994; Clarke et al. 1998b), was immuno¯uorescently localized in the bulge of the outgrowing
root hair and mainly accumulated in the apex, but also
throughout the cytoplasm of elongating maize root
hairs. In contrast, PIP2 immuno¯uorescence in Acanthamoeba, for instance, has been found to be mainly
limited to the plasma membrane (Bubb et al. 1998).
There is increasing evidence, however, that phosphatidylinositol 4-kinase is associated with cytoskeletal
elements in the cytoplasm and, furthermore, that the
lipid components of the phosphoinositide cycle may
directly be involved in regulation of the cytoskeletal
structure (Xu et al. 1992; Drobak et al. 1996; Clarke
et al. 1998b). This may also explain why PIP2 immuno¯uorescence was not detected in mature root hairs,
where the actin MF bundles are relatively stable.
However, the possibility of artifacts due to the ®xation
method can not be ruled out. In a recent paper, PIP2 was
reported to accumulate in the apical plasma membrane
of pollen tubes, where it may reorganize the MF system
441
(Kost et al. 1999). It was suggested that PIP2 may also
directly control exocytosis by recruiting required proteins or altering membrane lipid composition.
Mastoparan was used to interfere with pro®lin and
cytochalasin D was used to interfere with the actin
cytoskeleton. Treating elongating root hairs with
cytochalasin D disrupted the subapical actin MF bundles and disturbed the maintenance of the actin and
pro®lin caps. Apparently, this eliminated tip growth,
indicating a prominent role and intimate interaction of
actin and pro®lin in the apical dome. Mastoparan was
reported for instance to activate or stimulate phospholipases A2, C, and D, thereby a€ecting PIP2 turnover
and protein phosphorylation in animal and in plant cells
(e.g. Lassing and Lindberg 1985; Drobak and Watkins
1994; Clarke et al. 1998a; Munnik et al. 1998; Pingret
et al. 1998; Senda et al. 1998). Application of mastoparan resulted in a strong reduction or even a loss of
pro®lin ¯uorescence in the tip. In particular, it initiated
the transformation of the di€use actin cap into a ®ne,
netlike con®guration of actin MFs that merge into the
thick actin MF bundles of the root hair shaft. Despite
the fact that the e€ects of mastoparan are diverse and
not fully understood, it seems likely that it causes
alterations in PIP2 concentration. This might destabilize
the apical pro®lin-actin complexes to the extent that
polymerization of actin MFs is promoted, which in turn
would lead to the termination of tip growth.
In contrast to root hairs, tip-growing cell types that
actively adjust their direction of growth according to light
or gravity stimuli with internal perception mechanisms
such as gravity-sensing rhizoids and protonemata of the
green alga Chara, seem to require a highly organized
actin MF system in the apical dome (Braun and Wasteneys 1998). Both cell types exhibit an unique SpitzenkoÈrper composed of an aggregation of endoplasmic
reticulum membranes in its centre and an accumulation
of secretory vesicles organized by the extensive apical
actin MF system. Tip-growth of root hairs, however, is
not directed by internal orienting mechanisms, but can be
redirected by obstacles or by arti®cially generating an
asymmetrical calcium in¯ux across the root hair tip
(Bibikova et al. 1997). Having overcome the obstacles,
root hairs tend to return to their original growth
direction. A vesicle-rich apical dome containing a pool
of pro®lactin and a delicate network of short, probably
oligomeric actin MFs might be sucient or even advantageous for coordinating this mode of tip growth.
Our ®nding that pro®lin is speci®cally located at the
tip of growing root hairs is in contradiction to ®ndings
in other cell types where pro®lin was evenly distributed
throughout the cytoplasm (Mittermann et al. 1995;
Holzinger et al. 1997). Even in another tip-growing cell
type, the pollen tube, pro®lin was reported to be evenly
spread throughout the cytoplasm in chemically ®xed,
freeze-substituted and microinjected samples (Vidali and
Hepler 1997). The basis for this di€erence is unknown;
however, it is noteworthy that characean rhizoids and
protonemata which reorient their growth direction
according to external physical stimuli do not exhibit
apical pro®lin caps (data not shown). The growth
442
direction of root hairs is only transiently reoriented by
arti®cially setting a new lateral calcium gradient (Bibikova et al. 1997), and is continually being reset to the
original direction. Therefore, it seems likely that a tipgrowth mechanism coordinated by pro®lin-actin interactions is limited to the speci®c mode of root hair tip
growth.
It may deserve attention that pro®lin and PIP2 are
not only found in the apices of root hairs, but also
within the nuclei of trichoblasts and other root epidermal cells. This observation is in agreement with other
reports presenting speci®c localization of pro®lin
(Holzinger et al. 1997), PIP2 (Mazotti et al. 1995), the
actin depolymerizing factor ZmADF3 (Jiang et al. 1997)
and actin (Schindler and Jiang 1989) within the nucleus.
All these data and the ®nding that actin spikes appeared
within the nucleus of cytochalasin-treated root epidermal cells suggest that the G-actin binding protein
pro®lin plays a crucial role in tip growth of root hairs
and has a general physiological function in the transport
and dynamics of actin in plant cells.
The ZmPRO3 antibody and the recombinant pro®lin isoforms were
kindly provided by C.J. Staiger, Purdue University, Ind., USA. The
authors thank Simone Masberg for excellent technical assistance.
This work was supported by the AGRAVIS project of the Deutsches
Zentrum fuÈr Luft- und Raumfahrt (DLR) and the Ministerium fuÈr
Wissenschaft und Forschung, DuÈsseldorf, Germany.
References
Bibikova TN, Zhigilei A, Gilroy S (1997) Root hair growth in
Arabidopsis thaliana is directed by calcium and an endogenous
polarity. Planta 203: 495±505
Braun M, Wasteneys GO (1998) Distribution and dynamics of the
cytoskeleton in graviresponding protonemata and rhizoids of
characean algae: exclusion of microtubules and a convergence
of actin ®laments in the apex suggest an actin-mediated
gravitropism. Planta 205: 39±50
Bubb MR, Baines IC, Korn ED (1998) Localization of actobindin,
pro®lin I, pro®lin II, and phosphatidylinositol-4,5-bisphosphate
(PIP2) in Acanthamoeba castellanii. Cell Motil Cytoskel 39:
134±146
Buss F, Temm-Grove C, Henning S, Jockusch BM (1992)
Distribution of pro®lin in ®broblasts correlates with the
presence of highly dynamic actin ®laments. Cell Motil Cytoskel
22: 51±61
Cao LG, Babcock GG, Rubenstein PA, Wang YL (1992) E€ects of
pro®lin and pro®lactin on actin structure and function in living
cells. J Cell Biol 117: 1023±1029
CaÂrdenas L, Vidali L, Dominguez J, Perez H, Sanchez F, Hepler
PK, Quinto C (1998) Rearrangement of actin micro®laments in
plant root hairs responding to Rhizobium etli nodulation
signals. Plant Physiol 116: 871±877
Chaudhary A, Chen J, Gu Q, Witke W, Kwiatkowski DJ,
Prestwich GD (1998) Probing the phosphoinositide 4,5-bisphosphate binding site of human pro®lin I. Chem Biol 5: 273±
281
Clarke SR, Staiger CJ, Gibbon BC, Franklin-Tong VE (1998a)
A potential signaling role for pro®lin in pollen of Papaver
rhoeas. Plant Cell 10: 967±980
Clarke SR, Staiger CJ, Kovar DR, Franklin-Tong VE (1998b)
Phosphorylation of pollen pro®lins and inhibition by
phosphoinositides. Plant Cell 10: 967±980
M. Braun et al.: Actin, pro®lin and PIP2 in root hairs
De Corte V, Gettemans J, Vanderkerckhove J (1997) Phosphatidylinositol 4,5-bisphosphate speci®cally stimulates PP60c-src
catalyzed phosphorylation of gelsolin and related actin-binding
proteins. FEBS Lett 401: 191±196
De Ruijter NCA, Rook MB, Bisseling T, Emons AMC (1998)
Lipochito-oligosaccharides re-initiate root hair tip growth in
Vicia sativa with high calcium and spectrin-like antigen at the
tip. Plant J 13: 341±350
De Ruijter NCA, Emons AMC (1999) Actin-binding proteins in
plant cells. Plant Biol 1: 26±35
Drobak BK, Watkins PA (1994) Inositol(1,4,5)trisphosphate production in plant cells: stimulation by the venom peptides,
melittin and mastoparan. Biochem Biophys Res Commun 205:
739±745
Drobak BK, Watkins PAC, Valenta R, Dove SK, Lloyd CW,
Staiger CJ (1994) Inhibition of plasma membrane
phosphoinositide phospholipase C by the actin binding protein,
pro®lin. Plant J 6: 389±400
Emons AM (1987) The cytoskeleton and secretory vesicles in root
hairs of Equisetum and Limnobium and cytoplasmic streaming
in root hairs of Equisetum. Ann Bot 60: 625±632
Fechheimer M, Zigmond SH (1993) Focusing on unpolymerized
actin. J Cell Biol 123: 1±5
Gibbon BC, Ren H, Staiger CJ (1997) Characterization of maize
(Zea mays) pollen pro®lin function in vitro and in live cells.
Biochem J 327 (3): 909±915
Gibbon BC, Zonia LE, Kovar DR, Hussey PJ, Staiger CJ (1998)
Pollen tube function depends on interaction with proline-rich
motifs. Plant Cell 10: 981±994
Giehl K, Valenta R, Rothkegel M, Ronsiek M, Mannherz H-G,
Jockusch BM (1994) Interaction of plant pro®lin with mammalian actin. Eur J Biochem 226: 681±689
Hansson A, Skoglund G, Lassing I, Lindberg U, Ingelman-Sundberg
M (1988) Protein kinase C-dependent phosphorylation of pro®lin
is speci®cally stimulated by phosphatidylinositol bisphosphate
(PIP2). Biochem Biophys Res Comm 150: 526±531
Hartwig JH, Chambers KA, Hopeia KL, Kwiatkowski DJ (1989)
Association of pro®lin with ®lament-free regions of human
leucocytes and platelet membranes and reversible membrane
binding during platelet activation. J Cell Biol 109: 1571±1579
Heidstra R, Yang WC, Yalcin Y, Peck S, Emons AM, van
Kammen A, Bisseling T (1997) Ethylene provides positional
information in Nod factor-induced root hair tip growth in
Rhizobium-legume interaction. Development 124: 1781±1787
Holzinger A, Mittermann I, La€er S, Valenta R, Meindl U (1997)
Microinjection of pro®lins from di€erent sources into the green
alga Micrasterias causes transient inhibition of cell growth.
Protoplasma 199: 124±134
Jiang CJ, Weeds AG, Hussey PJ (1997). The maize actin-depolymerizing factor, ZmADF3, redistributes to the growing tip of
elongating root hairs and can be induced to translocate into the
nucleus with actin. Plant J 12: 1035±1043
Karakesisoglou KS, Schleicher M, Gibbon BC, Staiger CJ (1996)
Plant pro®lins rescue the aberrant phenotype of pro®linde®cient Dictyostelium cells. Cell Motil Cytoskel 34: 36±47
Kost B, Lemichez E, Spielhofer P, Hong Y, Tolias K, Carpenter C,
Chua N-H (1999) Rac homologues and compartmentalized
phosphatidyl 4,5-bisphosphate act in a common pathway to
regulate polar pollen tube growth. J Cell Biol 145: 317±330
Lassing I, Lindberg U (1985) Speci®c interaction between phosphatidylinositol 4,5-bisphosphate and pro®lactin. Nature 314: 472±
474
Machesky LM, Pollard TD (1993) Pro®lin as potential mediator of
membrane-cytoskeleton communication. Trends Cell Biol 3:
381±385
Mazotti G, Zini N, Rizzi E, Rizzoli R, Galanzi A, Ognibene A,
Santi S, Matteucci A, Martelli AM, Maraldi NM (1995)
Immunocytochemical detection of phosphatidylinositol 4,5bisphosphate localization sites within the nucleus. J Histochem
Cytochem 43: 181±191
M. Braun et al.: Actin, pro®lin and PIP2 in root hairs
Miller DD, Lancelle SA, Hepler PK (1997) Actin micro®laments
do not form a dense meshwork in Lilium longi¯orum pollen tube
tips. Protoplasma 195: 123±132
Miller DD, de Ruijter NCA, Bisseling T, Emons AMC (1999) The
role of actin in root hair morphogenesis: studies with lipochitooligosaccharide as a growth stimulator and cytochalasin as an
actin perturbing drug. Plant J 17: 141±154
Mittermann I, Swoboda I, Pierson E, Eller N, Kraft D, Valenta R,
Heberle-Bors E (1995) Molecular cloning and characterization of
pro®lin from tobacco (Nicotiana tabacum): Increased pro®lin
expression during pollen maturation. Plant Mol Biol 27: 137±146
Munnik T, van Himbergen JAJ, ter Riet B, Braun F-J, Irvine RF,
van den Ende H, Musgrave A (1998) Detailed analysis of the
turnover of polyphosphoinositides and phosphatic acid upon
activation of phospholipases C and D in Chlamydomonas cells
treated with non-permeabilizing concentrations of mastoparan.
Planta 207: 133±145
Perelroizen I, Didry D, Christensen H, Chua N-H, Carlier MF
(1996) Role of nucleotide exchange and hydrolysis in the
function of pro®lin in actin assembly. J Biol Chem 271: 12302±
12309
Pingret J-L, Journet E-P, Barker DG (1998) Rhizobium Nod factor
signaling: evidence for a G protein-mediated transduction
mechanism. Plant Cell 10: 659±671
Pollard TD, Cooper JA (1984) Quantitative analysis of the e€ect of
Acanthamoeba pro®lin on actin ®lament nucleation and elongation. Biochemistry 23: 6631±6641
Pring M, Weber A, Bubb MR (1992) Pro®lin-actin complexes
directly elongate actin ®laments at the barbed end. Biochemistry 31: 1827±1835
Ridge RW (1988) Freeze-substitution improves the ultrastructural
preservation of legume root hairs. Bot Mag Tokyo 101: 427±441
Schindler M, Jiang LW (1986) Nuclear actin and myosin as control
elements in nucleocytoplasmic transport. J Cell Biol 102: 859±862
SchluÈter K, Jockusch BM, Rothkegel M (1997) Pro®lins as regulators of actin dynamics. Biochim Biophys Acta 1359: 97±109
443
Seagull R, Heath I (1979) The e€ects of tannic acid on the in-vivo
preservation of micro®laments. Eur J Cell Biol 20: 185±188
Senda K, Doke N, Kawakita K (1998) E€ect of mastoparan on
phospholipase A2 activity in potato tubers treated with fungal
elicitor. Plant Cell Physiol 39: 1080±1086
Staiger CJ, Yuan M, Valenta R, Shaw PJ, Warn RM, Lloyd CW
(1994) Microinjected pro®lin a€ects cytoplasmic streaming in
plant cells by rapidly depolymerizing actin micro®laments. Curr
Biol 4: 215±219
Staiger CJ, Gibbon BC, Kovar DR, Zonia LE (1997) Pro®lin and
actin-depolymerizing factor: modulators of actin organization
in plants. Trends Plant Sci 2: 275±281
Sun H-Q, Kwiatkowska K, Yin HL (1995) Actin monomer binding
proteins. Curr Opin Cell Biol 7: 102±110
Tilney LG, Bonder EM, Coluccio LM, Moosecker MS (1983)
Actin from Thyone sperm assembles on only one end of an actin
®lament: a behavior regulated by pro®lin. J Cell Biol 97: 113±
124
Valster AH, Pierson ES, Valenta R, Hepler PK, Emons AMC
(1997) Probing the plant actin cytoskeleton during cytokinesis
and interphase by pro®lin microinjection. Plant Cell 9: 1815±
1824
Valenta R, Ferreira F, Grote M, Swoboda I, Vrtala S, Duchene M,
Deviller P, Meagher RB, McKinney E, Heberle-Bors E,
Kraft D, Scheiner O (1993) Identi®cation of pro®lin as an
actin binding protein in higher plants. J Biol Chem 268:
22777±22781
Vidali L, Hepler PK (1997) Characterization and localization of
pro®lin in pollen grains and tubes of Lilium longi¯orum. Cell
Motil Cytoskel 36: 323±338
Wymer CL, Bibikova TN, Gilroy S (1997) Cytoplasmic free
calcium distributions during the development of root hairs of
Arabidopsis thaliana. Plant J 12: 427±439
Xu P, Lloyd CW, Staiger CJ, Drobak BK (1992) Associaton of
phosphatidylinositol 4-kinase with the plant cytoskeleton. Plant
Cell 4: 941±951