Download Arabidopsis VILLIN4 is involved in root hair growth through

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Arabidopsis VILLIN4 is involved in root hair growth
through regulating actin organization in a Ca2+dependent manner
Yi Zhang*, Yingyu Xiao*, Fei Du, Lijuan Cao, Huaijian Dong and Haiyun Ren
Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education and College of Life Science, Beijing Normal University, Beijing
100875, China
Summary
Author for correspondence:
Haiyun Ren
Tel: +86 10 58806090
Email: [email protected]
Received: 18 October 2010
Accepted: 12 December 2010
New Phytologist (2011) 190: 667–682
doi: 10.1111/j.1469-8137.2010.03632.x
Key words: actin-binding protein, actin
bundle, actin cytoskeleton, cytoplasmic
streaming, villin.
• Villin is one of the major actin filament bundling proteins in plants. The function
of Arabidopsis VILLINs (AtVLNs) is still poorly understood in living cells. In this
report, the biochemical activity and cellular function of AtVLN4 were examined.
• The biochemical property of AtVLN4 was characterized by co-sedimentation
assays, fluorescence microscopy and spectroscopy of pyrene fluorescence. The in
vivo function of AtVLN4 was analysed by ectopically expressing it in tobacco pollen and examining the phenotypes of its T-DNA insertional plants.
• Recombinant AtVLN4 protein exhibited multiple activities on actin, including
actin filament bundling, calcium (Ca2+)-dependent filament severing and barbed
end capping. Expression of AtVLN4 in tobacco pollen induced the formation of
supernumerary actin cables and reduced pollen tube growth. Loss of function of
AtVLN4 resulted in slowing of root hair growth, alteration in cytoplasmic streaming routes and rate, and reduction of both axial and apical actin bundles.
• Our results demonstrated that AtVLN4 is involved in root hair growth through
regulating actin organization in a Ca2+-dependent manner.
Introduction
The actin cytoskeleton is a highly organized and dynamic
component of eukaryotic cells. It has crucial functions in
many physiological processes, including cell growth, cell
morphology and motility. Its dynamics and organization
are precisely controlled spatially and temporally by numerous actin-binding proteins (ABPs) (Dos Remedios et al.,
2003). In plant cells, actin filaments are often organized
into higher-order structures, such as bundles and cables,
which serve as tracks for cytoplasmic streaming and intracellular transport of organelles and vesicles (Shimmen et al.,
1995; Ye et al., 2009). Actin filament cross-linking and
bundling proteins are thought to be responsible for actin
bundle formation (Thomas et al., 2009).
Villin is one of the major proteins responsible for organizing actin filaments into bundles (Dos Remedios et al., 2003;
Thomas et al., 2009). It belongs to a multifunctional
superfamily of actin-binding proteins called the villin ⁄
*These authors contributed equally to this work.
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gelsolin ⁄ fragmin family (Friederich et al., 1999; Su et al.,
2007). This superfamily is a group of proteins sharing three
or six tandem 125–150-aa gelsolin homology domains, designated G1–G6. Gelsolin, the founding member of the
family, is composed of six gelsolin homology domains that
have distinct properties, endowing this protein with various
activities, including severing, capping and nucleating actin
filaments (Vandekerckhove, 1990; Burtnick et al., 1997; Su
et al., 2007). In addition to the gelsolin-like core domain,
typical villin contains an additional extension at its C-terminus, termed the headpiece (VHP), which allows each
molecule of villin to arrange actin filaments into bundles
(Friederich et al., 1990; Hartwig & Kwiatkowski, 1991).
Most, but not all, villins sever actin filaments and cap their
barbed ends at micromolar calcium (Ca2+) concentrations
(Hesterberg & Weber, 1983; Northrop et al., 1986; Janmey
& Matsudaira, 1988; Otto, 1994; Ferrary et al., 1999).
The first two villin homologs identified in plants were
P-135-ABP and P-115-ABP, which were isolated from lily
(Lilium longiflorum) pollen tubes by biochemical fractionation (Nakayasu et al., 1998; Yokota et al., 1998). Both of
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them are able to generate actin bundles with uniform polarity in a Ca2+ ⁄ calmodulin (CaM)-dependent manner in vitro
(Yokota et al., 1998, 2000, 2003; Yokota & Shimmen,
1999). Both P-135-ABP and P-115-ABP colocalize with
actin filament bundles in the transvacuolar strands and the
subcortical regions within the root hair cells of Hydrocharis
dubia (Tominaga et al., 2000; Yokota et al., 2003).
Microinjection of the antiserum against P-115-ABP or
P-135-ABP into living root hair cells disintegrates actin filament bundles, destroys transvacuolar strands and alters
cytoplasmic streaming routes, thus demonstrating that these
villin homologs are involved in maintenance of actin
bundles in root hairs (Tominaga et al., 2000; Yokota et al.,
2003). Moreover, biochemical experiments have shown that
P-135-ABP can form a complex with G-actin and accelerate
the polymerization and depolymerization of actin filaments
in the presence of Ca2+ ⁄ CaM (Yokota et al., 2005).
The Arabidopsis genome contains five villin-like genes
(AtVLN1-5) that are abundantly expressed in a wide range
of tissues, with elevated expression levels in certain types of
cells (Klahre et al., 2000). The full length of AtVLN3 and
the headpiece domains of AtVLN1 to AtVLN3, when fused
with green fluorescent protein (GFP), decorate actin filaments in plant and animal cells, thus demonstrating that
these AtVLNs bind to actin filaments in vivo (Klahre et al.,
2000). The biochemical activities and cellular functions of
several AtVLNs have recently been studied. Recombinant
AtVLN1 binds with high affinity to F-actins, bundles actin
filaments in a Ca2+- and CaM-insensitive manner, and protects actin filaments from ADF-mediated depolymerization,
but does not sever, cap or nucleate actin filaments (Huang
et al., 2005). AtVLN3 has overlapping and distinct activities with AtVLN1 (Khurana et al., 2010). It not only
bundles actin filaments in a Ca2+-independent manner,
but also severs actin filaments and bundles when Ca2+ is
elevated to micromolar levels (Khurana et al., 2010). Loss
of function of AtVLN5 sensitizes actin filaments in pollen
grains and tubes to latrunculin B, demonstrating that it is a
regulator of actin filament stability in pollen (Zhang et al.,
2010). As both AtVLN3 and AtVLN5 can sever actin
filaments at physiological Ca2+ concentrations in vitro,
they have been proposed to be involved in regulating
actin filament turnover in response to alteration of Ca2+
levels in plant cells, such as in pollen tubes (Khurana et al.,
2010; Staiger et al., 2010; Zhang et al., 2010). However,
more cytological evidence is still needed to support this
hypothesis.
We examined the biochemical properties of a villin isovariant, AtVLN4, and investigated its function within root
hairs of Arabidopsis. Our results demonstrate that AtVLN4
maintains all the typical activities of villin family members,
regulates the organization of long axial and short apical
actin bundles in root hairs, and is essential for normal root
hair growth and cytoplasmic streaming within root hairs.
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Materials and Methods
Plant materials and growth conditions
Arabidopsis thaliana (L.) Heynh. ecotype Columbia seeds
were surface sterilized and grown vertically on Murashige
and Skoog (MS) agar plates supplemented with 3.0%
sucrose in a controlled growth room with 16 h light : 8 h
dark cycles at 22C ± 2C.
Sequence alignment and phylogenetic analysis
Alignments were performed with CLUSTALX version 1.83
(http://bips.u-strasbg.fr/fr/Documentation/ClustalX/) using
default settings, and phylogenetic trees were constructed
using the neighbor-joining algorithm of MEGA version 4
(Tamura et al., 2007). A bootstrap test of the phylogeny
was performed with 1000 replications.
Plasmid construction and protein production
A full-length cDNA clone (U60239) for AtVLN4 was kindly
donated by the ABRC (Ohio State University, Columbus,
OH, USA). The coding sequence for AtVLN4 was subcloned into pBI121 under the control of a LAT52 promoter or
in frame with 6· His into pET-30a(+) (Novagen, Madison,
WI, USA), and confirmed by sequencing.
The error-free AtVLN4-pET-30a(+) construct was overexpressed in Escherichia coli strain BL21 (DE3). Cells were
grown to an OD600 of 0.6 at 37C in Luria–Bertani medium and induced with 0.5 mM isopropylthio-b-galactoside
at 22C for 12 h. Cultures were collected by centrifugation
and resuspended in binding buffer (400 mM NaCl,
40 mM phosphate buffer saline, pH 8.0) supplemented
with a 1 : 200 dilution of a stock solution of protease inhibitors (Ren et al., 1997). This was followed by purification
using a Ni-NTA His Bind Resin following the protocol in
the manufacturer’s manual (Novagen). The purified proteins were dialysed overnight against buffer G (2 mM TrisHCl, 200 lM CaCl2, 0.5 mM DTT, 200 lM ATP, pH
7.5) and stored in aliquots in liquid nitrogen. Protein
concentrations were determined with the Bradford reagent
(Bio-Rad), using BSA as a standard.
Actin was isolated from rabbit skeletal muscle acetone
powder using the method described by Pardee & Spudich
(1982) and labeled on Cys-374 with pyrene iodoacetamide
(Pollard, 1983) and Oregon Green 488 iodoacetamide
(Kuhn & Pollard, 2005).
Co-sedimentation assays
High-speed co-sedimentation assays were used to determine
the F-actin binding and depolymerizing activity of
AtVLN4. All proteins were preclarified at 200 000 g for
1 h before use. Actin was polymerized in the presence of
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1 · F buffer (buffer G with the addition of 50 mM KCl,
2.5 mM MgCl2 and 0.1 mM ATP) for 16 h at 4C.
AtVLN4 was incubated with 3.0 lM preformed F-actin in
the presence of various concentration of Ca2+ for 1 h at
room temperature (RT). To determine whether Ca2+ affects
the binding ability of AtVLN4 to actin filaments, AtVLN4
was incubated with phalloidin-stabilized-F-actin (3.0 lM)
in the presence of various concentrations of Ca2+. Free Ca2+
concentrations in the presence of ethylene glycol-bis(betaaminoethyl ether)-N,N,N ¢,N ¢-tetraacetic acid (EGTA)
were calculated using MAXC programs (available at http://
www.stanford.edu/~cpatton/maxc.html). According to our
calculations, 2.0 mM EGTA was equivalent to the volume
of 0.04 lM free Ca2+, 0.4 mM EGTA was equal to 0.5 lM
Ca2+, 0.2 mM EGTA was equal to 5 lM Ca2+ and
0.15 mM EGTA was equal to 50 lM Ca2+ in all of our
experimental systems. After centrifugation at 200 000 g for
1 h in a TLA-110 rotor (Beckman, Brea, CA, USA) at 4C,
equal amounts of supernatants and pellets were subjected to
12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant
Blue R 250 (DingGuo, Beijing, China). To determine Kd,
increasing amounts of AtVLN4 (0, 0.2, 0.4, 0.8, 1.6, 2.4 and
3.2 lM) were incubated with 5.0 lM preformed F-actin in
the presence of 2.0 mM EGTA for 1 h at RT. Samples were
analysed as described earlier, and the amount of AtVLN4 in
the pellets or supernatants was quantified using QUANTITY
ONE v4.6.5 software (Bio-Rad). The Kd value for AtVLN4
bound to F-actin was calculated by fitting the data of bound
protein vs free protein to a hyperbolic function using PRISM 5
software (GraphPad Software, Inc., San Diego, CA, USA).
Low-speed co-sedimentation assays were used to determine the actin bundling activity of AtVLN4. F-actin
(3.0 lM) was incubated with AtVLN4 in the presence of various concentration of Ca2+ for 30 min at RT. Samples were
centrifuged at 13 500 g for 30 min at 4C, analysed by SDSPAGE and the percentage of actin in each pellet was calculated. The experiments described earlier and the functional
assays described later were all repeated independently at least
three times using separate new preparations of each sample.
Actin filament depolymerization assays
Preassembled F-actin (3.0 lM, 50% pyrene-labeled) was
incubated with AtVLN4 in the presence of various concentration of Ca2+ for 5 min at RT and the sample was diluted
10-fold with 1· F buffer. The change of pyrene fluorescence intensity accompanying the F-actin depolymerization
was monitored for 600 s after dilution.
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for 5 min at RT. Then, 1.0 lM G-actin saturated with
4.0 lM human profilin I and one-tenth volume of 10· F
buffer were added to initiate actin polymerization at the
barbed end of actin filaments. The increase of pyrene fluorescence was monitored after the actin elongation was initiated.
Fluorescence microscopy visualization of actin
filaments and bundles
F-actin (3.0 lM) was incubated with 0.4 lM AtVLN4 in
the presence of 200 lM Ca2+ or 2.0 mM EGTA for 30 min
at RT and labeled with equimolar amount of Alexa 488-phalloidin (Molecular Probes, Eugene, Oregon, USA). F-actin
was diluted to 50 nM with 1· F buffer and observed using a
microscope (Carl Zeiss 200M, Germany) equipped with a
100 · ⁄ 1.5-numerical aperture Planapo objective. Digital
images were collected with an Axio CamMR charge-coupled
device camera using AxioVision software (Carl Zeiss).
Fluorescence microscopy to determine the severing
activity of AtVLN4
Oregon Green labeled actin (75% labeled) was polymerized
in the presence of 1· KMEI buffer (50 mM KCl, 1 mM
MgCl2, 1 mM EGTA and 10 mM imidazole, pH 7.0) at
RT for at least 1 h. Glass flow cells, with a capacity of c.
10 ll, were prepared each day as described in Kuhn &
Pollard (2005). Flow cells were blocked with two volumes of
1% BSA for at least 2 min, and washed with a fluorescence
buffer (10 mM imidazole pH 7.0, 50 mM KCl, 1 mM
MgCl2, 100 mM dithiothreitol (DTT), 0.2 mM ATP,
15 mM glucose, 20 lg ml)1 catalase, 100 lg ml)1 glucose
oxidase, 0.5% methylcellulose). Actin filaments (200 lM)
in the fluorescence buffer were introduced into the chambers
and allowed to settle for 5 min. Fluorescence buffer alone, or
a mixture of AtVLN4 at different concentrations of Ca2+,
was perfused into the flow cells. Actin filaments were
observed under an Observer Z1 microscope (Carl Zeiss)
equipped with an alphaPlanApo 100 · ⁄ 1.46-numerical
aperture oil objective. The time-course of actin filament
severing was photographed at 2 s or 5 s intervals using an
AxioCam Hsm camera (Carl Zeiss) and AxioVision software.
Exposure time was 900 ms. To quantify the severing activity
of AtVLN4, ‡ 20 filaments with lengths > 10 lm were chosen, and the average severing frequency was calculated from
the numbers of breaks, per unit of filament length, per unit
time (i.e. breaks lm)1 s)1) (Andrianantoandro & Pollard,
2006; Khurana et al., 2010; Zhang et al., 2010).
Seeded elongation assays
Particle bombardment-mediated transient expression
in tobacco pollen
Freshly prepared F-actin seeds (0.4 lM) were mixed with
AtVLN4 in the presence of various concentrations of Ca2+
Particle bombardment-mediated transient expression of
AtVLN4 in tobacco pollen was performed as described by
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Fu et al. (2001). Bombarded pollen grains were washed into
Petri dishes with 1.5 ml of germination medium (5 lM
CaCl2, 5 lM Ca(NO3)2, 1 mM MgSO4, 0.01% H3BO3,
15% sucrose, pH 6.5) and shaken at 80 rpm and 28C.
After incubation for 3 h, the pollen tubes were photographed to measure tube length. The untransformed pollen
tubes were used as a wild-type control.
Reverse-transcription polymerase chain reaction
(RT-PCR) analysis
Total RNA was isolated from 9-d-old Arabidopsis seedlings
using the plant RNA extraction kit according to the manufacturer’s instructions (AutoLab Biotechnology Co. Ltd.,
Beijing, China). Two micrograms of total RNA was added
to M-MLV reverse transcription kit (Promega Co.,
Madison, WI, USA) with Oligo-dT primers (Takara
Biotechnology (Dalian) Co., Ltd., Dalian, China) for the
synthesis of cDNA. Four microliters of the reaction product
was used as templates to amplify a 2131 bp fragment of
AtVLN4 in order to detect the expression level of AtVLN4
in wild type and mutants. Primers used were:
5¢-GCTATGTTTTCCAGTATTCTTATCCCG-3¢ (4-1f)
and 5¢-AAAAACTCAATCCTCAACTTCTGCAA-3¢ (4-2r).
ACTIN2 ⁄ 8 (forward primer 5¢-GGTAACATTGTGCTCAGTGGTGG-3¢ and reverse primer 5¢-AACGACCTTAATCTTCATGCTGC-3¢) genes were used as an
internal control. To detect the upstream product, a 176 bp
fragment of AtVLN4 was amplified using the following
primers: 5¢-AAGAATGAAGAAATAAACAGTTGATGGG-3¢ (v4uf) and 5¢-AGTGAAAGAGAGAGAATTGAATTGAGAG-3¢ (v4ur).
To further determine the expression level of AtVLN4 in
wild-type and mutant plants, quantitative real-time RTPCR was performed to amplify a 156 bp fragment using
the 7500 Real-Time PCR System (ABI, Carlsbad, CA,
USA) according to the manufacturer’s instructions. The
reactions were performed in a 20-ll volume containing
Power SYBR Green PCR Master Mix (ABI). Primers used
were: 5¢-GATTGTTTCTTCTTCAGGTCACAGGTT-3¢
(4-2f) and 4-2r. The level of eIF4A transcript (forward
primer 5¢-GGGTATCTATGCTTACGGTTTCG-3¢ and
reverse primer 5¢-CAGAGAACACTCCAACCTGAATC3¢) was amplified as an internal control.
To detect the expression of AtVLN4 in root hair cells, a
RT-PCR assay was performed according to the method
described by Zhou et al. (2005), with some modifications.
Root hairs were pulled using fine forceps and immersed
directly in lysis buffer containing 50 mM DTT and 10 units
ll)1 RNase Inhibitor (Takara). After freezing and thawing,
the root hairs were digested with RNase-free DNase
(TaKaRa), and the product was used as templates for the transcription of cDNA. Forty-five PCR cycles were performed
with the root hair cell-specific cDNA template and 30 cycles
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with the whole root cDNA template. AtVLN4-specific primers were designed to include introns so that the PCR products
amplified from cDNA (738 bp) could be distinguished from
PCR products amplified from genomic DNA (1400 bp).
The primers for AtVLN4 were 5¢-CCAGTCAAAGCGAGCCCG-3¢ and 5¢-GAAACCAAATTTCCTCAATTTGATACA-3¢. Two other genes, GLABRA2 and EXPANSINA7, were
used as controls (Lee & Cho, 2006; Lee et al., 2008).
Measurement of root hair length and growth rate
Root hairs were observed in 4-d-old seedlings. To measure
the length of root hairs, all the hairs in the same root region
(1.7–2.0 mm from the tip of each primary root) were measured using IMAGEJ software (http://rsb.info.nih.gov/ij). To
examine the growth rate, root hairs were photographed
twice with an interval of 2 h. The increase in length of each
root hair was measured and growth rate calculated.
Quantitative analysis of cytoplasmic streaming in root
hairs
Cytoplasmic streaming in root hairs was observed in 4-dold seedlings using an Observer D1 microscope (Carl Zeiss)
equipped with a 63 · ⁄ 1.25-numerical oil immersion
objective. Images were captured at 1-s intervals over a period of 2 min using an MRm Rev CCD camera (Carl Zeiss)
and AxioVision 4.2 software. Particles exhibiting continuous movement were selected at random to measure and
calculate the direction and velocity of particle movement.
Labeling of actin filaments in tobacco pollen and
Arabidopsis root hairs
Actin filaments in tobacco pollen tubes were labeled according
to the method described by Xiang et al. (2007). The stained
pollen was observed using a confocal laser scanning microscope (Olympus FV-300, Tokyo, Japan) mounted on an
inverted microscope (Olympus IX-70). Serial confocal optical sections were taken at a step size of 0.8 lm using
Olympus Fluoview 4.0 software.
Actin filaments in Arabidopsis root hair cells were labeled
as described previously (Miller et al., 1999; Ketelaar et al.,
2002, 2003), with some modifications. Four-d-old Arabidopsis
roots were prefixed for 2 min with 100 lM m-maleimido
benzoyl N-hydroxysuccinimide ester (Sigma) in 1% freshly
prepared paraformaldehyde and 0.025% glutaraldehyde in
actin-stabilizing buffer (ASB, 50 mM Pipes, 0.5 mM MgCl2,
0.5 mM CaCl2 and 37 mM KCl, pH 6.8).This was followed
by immersion in 200 lM ester, 2% paraformaldehyde and
0.05% glutaraldehyde in ASB for 20 min. Roots were then
fixed in a final concentration of 4% paraformaldehyde and
0.1% glutaraldehyde in ASB for 20 min. Roots were washed
three times in ASB, followed by permeabilizing with 100 lg
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ml)1 L-alpha-lysophosphatidylcholine (Sigma) in ASB for
5 min. Actin filaments were stained with 0.33 lM Alexa 488phalloidin (Molecular Probes) for 20 min, washed three times
in ASB and observed as already described.
Quantitative analysis of actin filament bundling in root
hairs
Skewness analysis was performed according to the method
described by Higaki et al. (2010). The z-series stacks of all
optical sections covering the whole root hair actin filament
images were filtered using Gaussian blur to reduce background noise and then skeletonized using the procedure of
ThinLine (a JAVA plug-in procedure; see Higaki et al.,
2010). The actin filament pixels were collected into a single
image using maximum-intensity projections and the skewness values were calculated.
To analyse the actin organization in different regions of
the root hairs, quantification of the fluorescence intensity of
actin cables was performed as described by Martin et al.
(2007), with small modifications. Lines were drawn perpendicularly to the long axis of the cell, across actin cables at
apical (5 lm and 10 lm from the tip), subapical (12 lm
and 15 lm from the tip) and shank (20 lm, 40 lm
and 60 lm from the tip) regions of the root hairs. The number of fluorescence intensity peaks, as well as the peak values,
along these lines was measured. The percentages of these
peak values at different intensity ranges were calculated.
Results
AtVLN4 binds to actin filaments
Based on phylogeny, plant villins were grouped into three
subclasses containing varying numbers of type 2 Ca2+-bind-
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ing sites (Choe et al., 2002) (see the Supporting
Information, Fig. S1). AtVLN4, along with AtVLN5,
belonged to clade III and possessed three type 2 Ca2+-binding sites in G1, G2 and G4 domains. Sequence alignment
further revealed that the overall structure and most of the
key amino acids were conserved in AtVLN4 (Fig. S2). The
results of phylogenetic analysis and sequence alignments
were consistent with the results of Huang et al. (2005) and
Khurana et al. (2010). These data imply that AtVLN4 possesses the general biochemical properties of villin family
members.
The ability of AtVLN4 to bind to F-actin was examined
using high-speed co-sedimentation assays. When AtVLN4
was centrifuged alone, little AtVLN4 was detected in the
pellet fraction (Fig. 1a, lanes 1 and 5). By contrast, in the
presence of 200 lM Ca2+ or 2.0 mM EGTA (Fig. 1a, lanes
3 and 7), a significant amount of AtVLN4 was found in the
pellet together with polymerized actin. These results indicate that AtVLN4 can bind to F-actin at both high and low
concentrations of Ca2+ in vitro. To determine the affinity of
AtVLN4 for binding to F-actin, increasing concentrations
of AtVLN4 were incubated with actin filaments in the presence of 2.0 mM EGTA. After centrifugation, the amount
of AtVLN4 in the supernatant and pellet fractions was
quantified by densitometric analysis. Fig. 1(b) shows a
representative experiment in which the concentration of
F-actin-bound AtVLN4 was plotted against the concentration of free AtVLN4, and the data were fitted with a
hyperbolic function. The average dissociation constant (Kd)
for AtVLN4 binding to F-actin was 0.24 ± 0.05 lM
(mean ± SD, n = 3). We also tested whether the binding
activity of AtVLN4 to actin filaments was affected by Ca2+.
As shown in Fig. 1(c), the percentage of AtVLN4 that
cosedimented with phalloidin-stabilized-actin filaments was
not statistically different (P > 0.05) at any of the Ca2+
Fig. 1 AtVLN4 binds to F-actin. (a) A high-speed co-sedimentation assay was used to determine the binding activity of AtVLN4 to F-actin.
Lanes 1 and 5, AtVLN4 alone in the pellet; lanes 2 and 6, AtVLN4 alone in the supernatant; lanes 3 and 7, AtVLN4 plus actin in the pellet;
lanes 4 and 8, AtVLN4 plus actin in the supernatant. Lanes 1–4 show the samples in the presence of 200 lM calcium (Ca2+), and lanes 5–8
show the samples in the presence of 2.0 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N‘,N‘-tetraacetic acid (EGTA). (b) Increasing
concentrations of AtVLN4 (0.2–3.2 lM) were incubated and co-sedimented with F-actin in the presence of 2.0 mM EGTA. The concentration
of F-actin-bound AtVLN4 was plotted against the concentration of free AtVLN4 and fitted with a hyperbolic function. For this representative
experiment, the Kd was 0.27 lM. (c) To determine whether Ca2+ affects the binding activity of AtVLN4 to actin filaments, AtVLN4 was
incubated with phalloidin-stabilized-F-actin (3.0 lM) in the presence of various concentrations of Ca2+ and the samples were subjected to high
speed co-sedimentation assays. Gels were analysed to determine the percentage of AtVLN4 in the pellet. Error bars indicate SE (n = 3).
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concentrations tested, indicating that AtVLN4 binds to Factin in a Ca2+-insensitive manner.
AtVLN4 bundles actin filaments
Low-speed co-sedimentation assays were performed to
examine the ability of AtVLN4 to bundle actin filaments.
Only small amounts of actin were detected in the pellet
fraction in the absence of AtVLN4 (Fig. 2a, lane 1). By
contrast, significantly more polymerized actin (P < 0.01)
sedimented in the presence of AtVLN4, and the amount
of actin in the pellet increased in proportion to AtVLN4
concentration (Fig. 2a, lanes 2–4; Fig. 2b). These data
suggest that AtVLN4 can bundle actin filaments in the
presence of 2.0 mM EGTA. To test whether the bundling
activity of AtVLN4 was Ca2+-dependent, low-speed
co-sedimentation assays were performed with fixed concentration of AtVLN4 in the presence of different
concentrations of Ca2+. The amount of sedimented actin
was significantly less at 50 lM (P = 0.006) and 200 lM
Ca2+ (P = 0.003) than at 0.04 lM Ca2+ (Fig. 2c). The
(a)
(d)
(b)
(e)
decreased amounts of actin in the pellet at high concentrations of Ca2+ could result from alterations in bundling,
severing or monomer-binding activities, or a combination
of these properties. It can be concluded, however, that
AtVLN4 bundles actin filaments at both high and low
concentrations of Ca2+.
The effect of AtVLN4 on actin filaments was directly
visualized using fluorescence microscopy. Actin filaments
were individually scattered in control samples (Fig. 2d). By
contrast, actin bundles were detected with the addition of
AtVLN4 (Fig. 2e,f). The bundles induced by AtVLN4 at
200 lM Ca2+ (Fig. 2e, mean length = 5.7 ± 4.1 lm, n =
94) were significantly shorter (P < 0.01) than those formed
in the presence of 2.0 mM EGTA (Fig. 2f, mean
length = 38.6 ± 15.8 lm, n = 37). Moreover, AtVLN4
significantly reduced the length of single actin filaments
(P < 0.01) in the presence of 200 lM Ca2+ (Fig. 2e, mean
length = 1.6 ± 0.6 lm, n = 100) compared with the control (Fig. 2d, mean length = 8.8 ± 4.3 lm, n = 100). The
reduction in filament length by AtVLN4 was dependent on
the concentration of Ca2+: reactions performed in the pres(c)
(f)
Fig. 2 AtVLN4 bundles actin filaments at different concentrations of calcium (Ca2+). (a) A low-speed co-sedimentation assay was used to
determine the bundling activity of AtVLN4 in the presence of 2.0 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N‘,N‘-tetraacetic acid
(EGTA). Lane 1, actin alone in the supernatant and pellet; lanes 2–4, actin plus 0.4 lM, 0.8 lM or 1.6 lM AtVLN4 in the supernatant and
pellet. (b) Statistical analysis of actin bundling activity of AtVLN4. Increasing concentrations of AtVLN4 (0.2–2.4 lM) were incubated with
F-actin in the presence of 2.0 mM EGTA and samples were subjected to low speed co-sedimentation assays. Gels were analysed to determine
the percentage of actin in the pellet. Error bars indicate SE (n = 3). (c) The effect of Ca2+ on bundling activity of AtVLN4 was determined by
low-speed co-sedimentation assays in the presence of different free Ca2+concentrations. Error bars indicate SE (n = 3). Asterisks represent
values that are statistically different (P < 0.05 by Student’s t-test) from the percentage of actin in the pellet at 0.04 lM Ca2+. (d–f) The ability
of AtVLN4 to generate actin bundles was visualized by fluorescence microscopy. F-actin (3.0 lM) was incubated with 0.4 lM AtVLN4 protein
in the presence of 200 lM Ca2+ (e) or 2.0 mM EGTA (f) at room temperature for 30 min and labeled with Alexa 488-phalloidin. A sample
with actin alone was used as a control (d). Bar, 5 lm.
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ence of 2.0 mM EGTA had a mean filament length of
8.3 ± 4.5 lm (Fig. 2f, n = 100). These observations confirm that AtVLN4 generates actin bundles at both high and
low concentrations of Ca2+.
AtVLN4 severs actin filaments in a Ca2+-dependent
manner
High-speed co-sedimentation assays were performed to
examine the effect of AtVLN4 on actin filaments. In the
presence of 200 lM Ca2+, AtVLN4 retained substantially
more actin (P < 0.05) in the supernatant (Fig. S3a, lanes
2–4; Fig. S3b) than did the controls (Fig. S3a, lane 1;
Fig. S3b). However, when the Ca2+ concentration was chelated to 0.5 lM or 0.04 lM, the amount of actin in the
supernatant was not significant (P > 0.05) compared with
the actin alone control (Fig. S3b,c). AtVLN4 also promoted dilution-mediated actin filament depolymerization
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in a Ca2+-dependent manner (Fig. S3d,e). These data suggest that AtVLN4 can depolymerize or sever actin filaments
in a Ca2+-dependent manner. Time-lapse fluorescence
microscopy was employed to directly determine whether
AtVLN4 severs actin filaments. Few breaks were observed
in the control sample (Video S1). By contrast, AtVLN4
induced breaks along actin filaments over time (Fig. 3a;
Video S3). The number of breaks per unit of filament
length per second (breaks lm)1 s)1) was calculated as the
‘severing frequency’ to quantify the severing activity of
AtVLN4 (Andrianantoandro & Pollard, 2006; Khurana
et al., 2010; Zhang et al., 2010). AtVLN4 significantly
raised the severing frequency over that of the actin alone
control (P < 0.01). The severing frequency increased in
proportion to AtVLN4 concentration (Fig. 3b; Videos S1–
S5). The severing was markedly faster at concentrations
> 10 nM, making it difficult to assess quantitatively. To
determine whether the severing activity of AtVLN4 was
Fig. 3 AtVLN4 severs actin filaments in a calcium (Ca2+)-dependent manner. (a) Severing activity of AtVLN4 is directly observed by
fluorescence microscopy. Oregon Green-labeled actin filaments (200 lM) were introduced into a flow cell, and 1.0 nM AtVLN4 in the
presence of 200 lM Ca2+ was applied at time zero. Actin filaments showed breaks (arrows) as time elapsed. The time in seconds following
addition of AtVLN4 is shown in the bottom right corner of each panel. See also the Supporting Information, Video S3. Bar, 10 lm. (b)
Statistical analysis of severing activity. Different concentrations of AtVLN4 were perfused into the flow cells containing Oregon Green actin
filaments in the presence of 200 lM Ca2+. Severing frequency was calculated as the number of breaks per unit of filament length, per unit
time. At least 20 filaments for each experimental treatment were counted. Error bars represent SE (n = 3). Asterisks represent values that are
statistically different (P < 0.05 by Student’s t-test) from that of the control. (c) The severing activity of AtVLN4 is Ca2+ dependent. AtVLN4
(5.0 nM) in the presence of various concentrations of Ca2+ was introduced into flow cells containing Oregon Green actin filaments. At least 20
filaments for each experimental treatment were selected to calculate the average severing frequency. Error bars represent SE (n = 3). Asterisks
represent values that are statistically different (P < 0.05 by Student’s t-test) from that of the control.
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Ca2+-dependent, similar assays were performed with fixed
concentrations of AtVLN4 in the presence of different
concentrations of Ca2+. As shown in Fig. 3(c), the severing
frequency reduced with the decreasing of Ca2+ concentration (Videos S5–S9). When the Ca2+ concentration was 0
or 0.5 lM, the average severing frequency was not statistically different (P = 0.86) from that of the actin alone
control. These data indicate that AtVLN4 can sever actin
filaments in a Ca2+-dependent manner.
After severing, villin ⁄ gelsolin ⁄ fragmin family member
remains attached to the barbed ends of the actin filaments
as a cap, thereby preventing actin fragments from reannealing.
Seeded elongation assays were also employed to determine
the capping activity of AtVLN4. The results demonstrate
that AtVLN4 caps the barbed ends of actin filaments in a
Ca2+-dependent manner (Fig. S4).
AtVLN4 colocalizes with actin filaments, induces the
formation of actin cables and decreases tube growth
in tobacco pollen
We next sought to determine the effect of AtVLN4 overexpression on actin organization in plant cells. Full-length
AtVLN4 fused with GFP was expressed under the control
of a pollen-specific promoter Lat52 in tobacco pollen. The
GFP fluorescence was distributed evenly throughout the
whole cell (Fig. S5a,b). By contrast, AtVLN4-GFP was
detected associated with a longitudinally arrayed filamentous network (Figs 4a, S5c,d). Alexa 568-phalloidin
labeling of the transformed cells revealed that AtVLN4
colocalized with actin filaments, especially the thick actin
cables, in the apical, subapical, shank and basal regions of
the pollen tube and the pollen grain (Fig. 4a). When treated
with Latrunculin B, an inhibitor of actin polymerization
that causes the actin network to disassemble (Morton et al.,
2000), both the AtVLN4-GFP organization and actin
networks were disrupted (Fig. 4a).
To examine the effect of AtVLN4 overexpression on the
actin cytoskeleton, we compared the actin organizations of
wild type, GFP and AtVLN4-GFP transformed pollen
tubes. As shown in Fig. 4(b), confocal imaging of Alexa
568-phalloidin fluorescence revealed that the actin filament
network in wild-type pollen tubes consisted of thick and
fine, often longitudinally oriented, cables. Overexpression
of GFP did not affect actin organization (Fig. 4b). By contrast, AtVLN4-GFP tubes showed more thick actin cables
than did wild-type or GFP cells (Fig. 4b); indicating that
AtVLN4 induces the formation of more actin cables in
tobacco pollen. Moreover, pollen tube growth was greatly
altered following transformation with AtVLN4. As shown
in Fig. 4(c), the length of pollen tubes growing for 3 h was
significantly reduced when AtVLN4 was overexpressed. The
average length, 292.5 ± 90.9 lm (n > 150), was significantly shorter than that of the untransformed tubes (350.6 ±
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133.2 lm, n > 150; P < 0.01) or the tubes transformed
with GFP (340.6 ± 100.1 lm, n > 150; P < 0.01).
T-DNA insertion mutants of the AtVLN4 gene exhibit
a short-root-hair phenotype
To gain an insight into the possible functions of AtVLN4
in Arabidopsis, we used RT-PCR analysis to investigate its
expression pattern within different tissues. As shown in
Fig. S6(a), AtVLN4 had higher expression levels in roots
than in aerial tissues. The data from the Affymetrix ATH1
GeneChip arrays in the Genevestigator database further
indicate that AtVLN4 (At4g30160) is more abundant in the
root hair zone than in the root tip and elongation zone
(Zimmermann et al., 2004) (Fig. S6b). The root map for
AtVLN4 expression generated from the AREX database
suggests more directly that AtVLN4 might be expressed in
root hair cells (Birnbaum et al., 2003; Brady et al., 2007;
Cartwright et al., 2009) (Fig. S6c). To verify this, the
expression of AtVLN4 in root hair cells was examined. As
shown in Fig. 5(a), the AtVLN4 transcript was present in
root hair cells.
To analyse the function of AtVLN4 in Arabidopsis, we
obtained two T-DNA insertion lines, SALK_049058
(atvln4-1) and SAIL_517_A03 (atvln4-2), from the ABRC
and isolated homozygous plants (Fig. S7a,b). Sequencing
analysis of the T-DNA borders revealed that atvln4-1 and
atvln4-2 have T-DNA insertion at 88 bp downstream of the
start of the 10th intron and at 416 bp downstream of the
start of 3¢-UTR, respectively (Fig. 5b). In RT-PCR analysis,
using total RNA prepared from whole seedlings, both the TDNA insertion lines prevented accumulation of full-length
transcripts in the mutational plants (Fig. 5(d), inset), which
was further confirmed by quantitative real-time RT-PCR
(Fig. S7d). Neither of the mutant lines showed phenotypic
changes in their primary root length (Fig. S8). However,
both the mutant lines had shorter root hairs than those of
wild-type plants (Fig. 5c). As shown in Fig. 5(d), the length
of root hairs was significantly reduced in atvln4-1 (167.6 ±
25.0 lm; P < 0.01) and atvln4-2 (201.9 ± 33.1 lm;
P < 0.01) plants compared with wild-type plants (336.7 ±
43.1 lm). The growth rate of root hairs also significantly
decreased in atvln4-1 (0.75 ± 0.34 lm min)1; P < 0.01)
and atvln4-2 (0.77 ± 0.32 lm min)1; P < 0.01) plants
compared with wild-type plants (1.03 ± 0.34 lm min)1).
These data suggest that AtVLN4 is required for normal root
hair growth.
Direction and velocity of cytoplasmic streaming are
altered in atvln4 root hairs
We then observed cytoplasmic streaming within atvln4 root
hairs. As shown in Video S10, wild-type root hairs presented
a known pattern of reverse fountain streaming: particles rap-
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(a)
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AtVLN4-GFP
F-actin
(b)
Merge
(c)
Fig. 4 AtVLN4 colocalizes with actin filaments, induces the formation of actin cables and slows tube growth of tobacco pollen. (a) Tobacco
pollen was transformed with Lat52:AtVLN4-GFP plasmid (3.0 lg) by microprojectile bombardment, and stained with Alexa 568-phaloidin (red
signal). AtVLN4-GFP and actin filaments were colocalized in the pollen grain (the top panel) and the pollen tube (the middle panel), as judged
by the presence of yellow filaments in the merged images. Inset showed the localization of AtVLN4-GFP and actin filaments in the tip region
of pollen tube. When treated with 100 nM Latrunculin B (LatB), both the structures of AtVLN4-GFP and actin filaments were disrupted (the
bottom panel). Bars, 10 lm. (b) Comparison of actin organization in untransformed (WT), GFP and AtVLN4-GFP (AtVLN4) transformed
tobacco pollen. Staining conditions and imaging parameters were identical between pollen samples. Images shown are z-series stacks of all
optical sections. For each group, > 10 pollen tubes were observed and showed similar staining patterns. Bar, 10 lm. (c) Length of pollen tubes
(mean ± SE, n > 150) after germination in culture medium for 3 h. Asterisk represents values that are statistically different (P < 0.05 by
Student’s t-test) from that of the control. These data were obtained from three independent experiments.
idly moved to the apex along the cortex and moved back in
the center of the cell after reaching the tip region. However,
the pattern of cytoplasmic streaming appeared disturbed
and irregular in atvln4-1 (Video S11) and atvln4-2 (Video
S12) root hairs. Cytoplasmic streaming routes became transverse to the axis of root hair growth (Fig. 6a). In order to
quantify the alteration in streaming routes, we measured the
angles between the tracks of particle movement and the axis
of root hair growth. As shown in Fig. 6(b), the tracks of particles were always longitudinal (angle from 0 to 10) or
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slightly oblique (angle from 10 to 30) to the growth axis in
wild-type root hairs. By contrast, c. 50% particles moved in
a severe oblique (angle from 30 to 80) or even transverse
direction (angle from 80 to 90) in atvln4 root hairs. We
also measured the velocity of cytoplasmic streaming in root
hairs for each genotype. As shown in Fig. 6(c), the average
velocity of cytoplasmic streaming was significantly reduced
in atvln4-1 (0.82 ± 0.23 lm s)1, n = 40; P < 0.01) and
atvln4-2 (0.98 ± 0.20 lm s)1, n = 40; P < 0.01) root hairs
compared with that of wild type root hairs (1.36 ±
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Fig. 5 AtVLN4 is expressed in root hairs and its T-DNA insertion mutants exhibit a short-root-hair phenotype. (a) Reverse-transcription
polymerase chain reaction (RT-PCR) analysis of AtVLN4 transcript in Arabidopsis root hair cells. EXPANSINA7 (E7) and GLABRA2 (GL2)
were used as controls for hair cell-specific and nonhair-cell-specific amplification. Gene-specific primer sets were designed to include introns
to distinguish amplification from cDNA vs genomic DNA. (b) Location of T-DNA insertions. The structure of AtVLN4 is shown schematically,
with coding (gray) and non-coding (white) regions. Closed triangles indicate the sites of T-DNA insertion in the mutant lines. Open triangles
indicate the positions of primers used in the RT-PCR analysis. (c) Root hair phenotype of atvln4 mutants. Bar, 0.5 mm. (d) Root hair length
(closed columns, mean ± SE, n > 500 from at least 30 individual roots) and growth rate (open columns, mean ± SE, n > 60 from at least 10
individual roots) of wild-type (WT) plants and atvln4 mutants. Asterisks represent values that are statistically different (P < 0.01 by Student’s
t-test) from that of the control. These data were obtained from three independent experiments. Inset: RT-PCR analysis of AtVLN4 transcript in
wild-type and T-DNA insertional mutants. The ACTIN2 ⁄ 8 gene was amplified as an internal control.
0.24 lm s)1, n = 40). Together, these data suggest that
AtVLN4 is essential for normal cytoplasmic streaming in
root hairs.
Actin organization is affected in atvln4 root hairs
To visualize actin organization, root hairs of wild-type and
atvln4-1 plants were subjected to actin staining with Alexa488 phalloidin. As shown in Fig. 7(a), actin cables were
arranged longitudinally in the shank of the wild-type root
hair, and some short actin filaments were present in the tip.
However, the prominent long actin cables were almost completely absent in the shank of atvln4 root hairs and were
replaced by some discontinuous short cables (Fig. 7b). Two
methods were employed to quantitatively evaluate actin
organization in root hairs. First, a statistical parameter called
skewness, which has been successfully applied to examine
the extent of actin filament bundling in living guard cells of
Arabidopsis (Higaki et al., 2010), was calculated for wildtype and atvln4 root hairs. The skewness value became
higher when high fluorescence intensity pixel numbers were
increased by actin filament bundling. As shown in Fig. 7(e),
the average skewness value was significantly decreased for
atvln4 root hairs (1.20 ± 0.21, n = 14; P < 0.01) compared
with that of wild-type root hairs (1.98 ± 0.62, n = 14).
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These data indicate that fewer actin polymers were organized into high-order cables in atvln4 root hairs. Second,
the fluorescence intensities of actin cables were further analysed in different regions of root hair cells. Lines were drawn
perpendicularly to the long axis of the root hair cell and the
number of fluorescence intensity peaks, as well as the peak
values, along the lines were measured. As shown in Fig. 7(f–
h), the histogram of fluorescence intensity distributions for
actin cables showed that the percentages of actin cables with
low fluorescence intensity (< 600 au) significantly increased
(P < 0.01 by v2-test) in atvln4 root hairs compared with
those in wild-type hairs. These data suggest that atvln4 root
hairs had consistently fewer high-order actin cables in the
shank, subapical and apical regions than did the wild-type
root hairs. Collectively, these data indicate that AtVLN4 is
responsible not only for the formation and ⁄ or maintenance
of long axial actin cables in the shank region, but also for the
short actin cables in the subapical and apical regions of
Arabidopsis root hairs.
Discussion
In this paper, we have characterized the biochemical activities of AtVLN4 in vitro and analysed its function in plant
cells. Sequence alignment reveals that the key amino acids
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Fig. 6 Direction and velocity of cytoplasmic streaming are altered in atvln4 root hairs. (a) Cytoplasmic streaming routes in wild-type and
atvln4 root hairs. The tracks of several particles were followed over a few seconds within root hairs. The arrowhead indicates the current
position of a single particle, while the recent position of the same particle is recorded by colored dots. The time in seconds following imaging is
shown in the top right corner of each panel. See also the Supporting Information, Videos S10–S12. Bar, 10 lm. (b) Quantitative analysis of
streaming direction in wild-type and atvln4 root hairs. The angles between the tracks of particle movement and the axis of root hair growth
were measured and grouped into four types: longitudinal (angles from 0 to 10), oblique (angles from 10 to 30), severe oblique (angles
from 30 to 80) and transverse (angles from 80 to 90). The percentage of particles within each group was analysed and presented. Forty
cytoplasmic streaming particles from several independent root hairs were measured for each genotype. (c) Velocity of cytoplasmic streaming
(mean ± SE, n = 40) in wild-type and atvln4 root hairs. Asterisks represent values that are statistically different (P < 0.01 by Student’s t-test)
from the velocity of cytoplasmic streaming in wild-type root hairs. These data were obtained from three independent experiments.
are conserved in AtVLN4 (Fig. S2), implying that AtVLN4
maintains the general biochemical activities of villin family
members. Indeed, AtVLN4 exhibits multiple effects on
actin filaments. First, low-speed co-sedimentation assays
and direct visualization of actin filaments by fluorescence
microscopy show that AtVLN4 efficiently bundles actin filaments at both low and high concentrations of Ca2+ (ranging
from 0.04 lM to 200 lM), although bundles are much
shorter at 200 lM Ca2+ than at 0.04 lM Ca2+. These data
imply that AtVLN4 may contribute to the generation of
long and short actin filament bundles under the regulation
of Ca2+ in the plant cells. Second, time-lapse fluorescence
microscopy using Oregon Green actin filaments reveals that
AtVLN4 exhibited a dose-dependent increase in severing
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frequency with increasing Ca2+. This has similarity to the
behavior of AtVLN3 (Khurana et al., 2010) and AtVLN5
(Zhang et al., 2010). AtVLN4, as well as AtVLN3 and
AtVLN5, severs actin filaments at micromolar Ca2+ concentrations, making it a potential candidate for actin filament
severing in plant cells. Finally, seeded actin elongation
assays show that AtVLN4 can effectively cap the barbed
ends of actin filaments in a Ca2+-dependent manner, with
increased concentration of Ca2+ enhancing the capping
activity. Thus, as stated above, AtVLN4 retains all the activities of villin family members, and is similar to human
villin (Walsh et al., 1984) and AtVLN5 (Zhang et al.,
2010), but is markedly different from AtVLN1 (Huang
et al., 2005).
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(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Fig. 7 Actin organization is affected in atvln4 root hairs. (a–d) The actin cytoskeleton of a wild-type root hair (a) and an atvln4-1 root hair (b)
was visualized by confocal laser scanning microscopy after staining with Alexa 488-phalloidin. (c) and (d) show the corresponding bright-field
images for (a) and (b) root hairs, respectively. Staining conditions and imaging parameters were identical between cells. Images shown are zseries stacks of all optical sections. For each line, > 20 root hairs were observed and showed similar staining patterns. Bar, 10 lm. (e) The actin
cable level was substantially decreased in atvln4 root hairs based on skewness analysis. The skewness value of each root hair analysed is shown
(n = 14). Dashed lines indicate the mean value of each group. Asterisks represent values that are statistically different (P < 0.01 by Student’s ttest) between the wild type and the atvln4 line. (f–h) To quantify the fluorescence intensity of actin cables in different regions of wild-type and
atvln4 root hair cells, we measured the peaks of the fluorescence profiles along lines drawn across actin cables. A histogram of the peak values
in the shank (f), subapical (g) and apical (h) regions of the root hairs is shown. Error bars indicate ± SE. Wild type (n = 25), closed bars; atvln4
(n = 20), open bars. These data were obtained from three independent experiments. Wild type and atvln4 root hairs show statistically
different values in the shank (P < 0.01 by v2-test), subapical (P < 0.01 by v2-test) and apical (P < 0.01 by v2-test) regions.
Root hair cells are specialized root epidermal cells with
tubular extensions, whose development, together with pollen tubes, serves as an attractive working model for
investigation of the highly complex tip growth of plant
cells. In these cells, actin filament cables orientated parallel
to the long axis of the tube are well characterized, and are
believed to play a prominent role as tracks for the intracellular transport of organelles and vesicles (Miller et al.,
1999; Sheahan et al., 2004; Lovy-Wheeler et al., 2005; Ye
et al., 2009). Villin, one of the major actin bundling
proteins in animal cells, has been indicated to be involved
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in organizing and maintaining actin bundles in plant cells.
Lily villins colocalize with actin cables in pollen tubes and
root hairs and are responsible for bundle formation and
maintenance in these cells (Yokota et al., 1998, 2003;
Tominaga et al., 2000). AtVLN5 loss-of-function retards
pollen tube growth and sensitizes actin filaments in pollen
grains and tubes to Latrunculin B, demonstrating its role in
regulating actin filament stability in pollen (Zhang et al.,
2010). Injection of an anti-lily villin antibody into growing
root hairs of Arabidopsis leads to actin filament unbundling
(Ketelaar et al., 2002), implying that AtVLNs may also
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participate in actin bundle formation and maintenance in
root hairs. AtVLN4 bundles actin filaments in vitro and
ectopic expression of AtVLN4 in tobacco pollen causes the
formation of excessive actin cables. AtVLN4 is expressed in
root hairs. Moreover, knock-out of AtVLN4 eliminates the
long axial actin cables and causes the formation and ⁄ or existence of fewer actin cables in root hairs. This alters the
direction and velocity of cytoplasmic streaming in root hairs
and leads to reduced root hair growth. These data demonstrate that AtVLN4 is responsible for the formation and ⁄ or
maintenance of long axial actin cables in the shank regions
of root hairs. Overexpression and loss of function of
AtVLN4 have contrary effects on actin organization, but
both result in impaired growth rate of pollen tubes and root
hairs, suggesting that actin organization is precisely controlled and regulated in plant cells. Our results also provide
evidence that intact, longitudinal actin cables are essential
for cytoplasmic streaming in root hairs and that AtVLN4 is
a significant regulator of this process. All the AtVLNs studied, including AtVLN1 (Huang et al., 2005), AtVLN3
(Khurana et al., 2010) and AtVLN5 (Zhang et al., 2010),
efficiently bundle actin filaments in vitro. These villin isovariants may cooperate to generate actin bundles within
plant cells. AtVLN1 and AtVLN3 coexist in many cell types
and have overlapping and distinct activities in actin bundle
formation and turnover (Khurana et al., 2010). In addition
to the villin proteins, several other actin-bundling proteins
have been identified, including formin (Cheung & Wu,
2004; Ye et al., 2009), fimbrin (Kovar et al., 2000) and
LIM proteins (Thomas et al., 2006; Papuga et al., 2010).
There may be cooperative action among these actin bundling proteins to elaborate actin bundles in plant cells.
Similar to pollen tubes, the actin cytoskeleton is subjected
to significant rearrangement in the subapical regions and
tips of root hairs. The long actin bundles in the shank flare
out into short and fine bundles, whose precise conformation
has not yet been clearly defined, at the subapical regions of
root hairs (Miller et al., 1999; Ketelaar et al., 2002, 2003).
Debate surrounds the presence of filamentous actin in the
extreme apical areas of growing root hairs (Baluska et al.,
2000; Ketelaar et al., 2003; Sheahan et al., 2004; Wang
et al., 2004; Voigt et al., 2005). Root hairs, as well as pollen
tubes, have an oscillatory, tip-high Ca2+ gradient that is
considered responsible for regulating and modulating the
dynamics and arrangement of the actin cytoskeleton by the
activation ⁄ inactivation of Ca2+-dependent ABPs (Ren &
Xiang, 2007). With evidence of P-135-ABP accelerating the
polymerization and depolymerization of actin in a Ca2+dependent manner, as well as AtVLN3 and AtVLN5 severing actin filaments in the physiological range of Ca2+ (1–
10 lM), villins have been implicated to be Ca2+-regulated
proteins involved in modifying actin organization and
dynamics in the apical regions of root hairs and pollen tubes
(Yokota et al., 2005; Khurana et al., 2010; Staiger et al.,
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2010; Zhang et al., 2010). AtVLN4 possesses severing
activities on actin filaments at physiological Ca2+ levels.
Moreover, AtVLN4 caps the barbed ends of actin filaments
in a Ca2+-dependent manner. Thus, it is plausible to predict
that AtVLN4 may participate in regulating actin organization in the regions close to the tips of root hairs. Indeed,
loss of function of AtVLN4 results in fewer actin cables in
the subapical and apical regions of root hairs. Along with
the result that AtVLN4 generates relatively short actin bundles at high concentrations of Ca2+in vitro, we provide
evidence that AtVLN4 is involved in organizing actin filaments into fine and short bundles, probably through its
actin filament bundling, severing and ⁄ or capping activities,
in the subapical and apical regions of root hairs. Recently,
Staiger et al. (2009) directly examined the behavior of cortical actin in epidermal cells and revealed that actin filament
dynamics are dominated by rapid growth and severing
activity. Further work directly measuring dynamic parameters of the actin cytoskeleton in atvln4 root hairs is
required to verify and illustrate whether and how villin
isoforms regulate actin dynamics in the apical regions in
response to tip-high Ca2+ fluxes.
Acknowledgements
We thank Dr David Kovar and Yujie Li (University of
Chicago, IL, USA) for their helpful suggestions on actin filament severing assays. We also thank Dr Jinxing Lin and
Dr Yinglang Wan (Institute of Botany, Chinese Academy
of Sciences) for their friendly help with microscope use.
This work was supported by the National Basic Research
Program of China (2007CB108700), the National Natural
Science Foundation of China (30630005, 30870211,
30970174) and the Chinese Transgenic Project (2009
ZX08009-059B) to H.R.
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Supporting Information
Additional supporting information may be found in the
online version of this article.
Fig. S1 Phylogenetic analysis of villins from angiosperms.
Fig. S2 Multiple sequence alignment of plant villin proteins.
Fig. S3 AtVLN4 severs or depolymerizes actin filaments in
a calcium (Ca2+)-dependent manner.
Fig. S4 AtVLN4 inhibits actin filament elongation from
barbed ends in a calcium (Ca2+)-dependent manner.
Fig. S5 Subcellular localization of GFP, AtVLN4-GFP and
GFP-fABD2 in tobacco pollen.
Fig. S6 AtVLN4 is abundant in the Arabidopsis root hair
zone.
Fig. S7 Molecular analysis of AtVLN4 T-DNA insertion
lines.
Fig. S8 Comparison of primary root length (mean ± SE,
n > 20) in wild-type and atvln4 plants.
Video S1 Time-lapse fluorescence microscopy series of
200 lM Oregon Green-actin filaments treated with fluorescence buffer only.
Video S2 Actin filaments severed by 0.5 nM AtVLN4 in
the presence of 200 lM calcium (Ca2+).
Video S3 Actin filaments severed by 1 nM AtVLN4 in the
presence of 200 lM calcium (Ca2+).
Video S4 Actin filaments severed by 2.5 nM AtVLN4 in
the presence of 200 lM calcium (Ca2+).
Video S5 Actin filaments severed by 5 nM AtVLN4 in the
presence of 200 lM calcium (Ca2+).
Video S6 Actin filaments show minimal breakage when
treated with 5 nM AtVLN4 in the absence of calcium
(Ca2+).
Video S7 Actin filaments show minimal breakage when
treated with 5 nM AtVLN4 in the presence of 0.5 lM calcium (Ca2+).
Video S8 Actin filaments severed by 5 nM AtVLN4 in the
presence of 5 lM calcium (Ca2+).
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Video S9 Actin filaments severed by 5 nM AtVLN4 in the
presence of 50 lM calcium (Ca2+).
Video S10 This movie corresponds to the pattern of cytoplasmic streaming of a wild-type root hair (shown in
Fig. 6a, left panels).
Video S11 This movie corresponds to the pattern of cytoplasmic streaming of an atvln4-1 root hair (shown in
Fig. 6a, middle panels).
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Video S12 This movie corresponds to the pattern of cytoplasmic streaming of an atvln4-2 root hair (shown in
Fig. 6a, right panels).
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting information
supplied by the authors. Any queries (other than missing
material) should be directed to the New Phytologist Central
Office.
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