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The Plant Journal (2010) 62, 925–935 doi: 10.1111/j.1365-313X.2010.04210.x

A role for ABIL3 in plant cell morphogenesis

Cordula I. Jörgens, Nora Grünewald, Martin Hülskamp* and Joachim F. Uhrig*
Botanical Institute III, University of Köln, Gyrhofstr. 15, 50931 Köln, Germany

Received 15 January 2010; accepted 4 March 2010; published online 27 April 2010.
*
For correspondence (fax +49 221 470 5062; e-mail joachim.uhrig@uni-koeln.de; martin.huelskamp@uni-koeln.de).

SUMMARY
Actin nucleation facilitated by the ARP2/3 complex plays a central role in plant cell shape development. The
molecular characterization of the distorted class of trichome mutants has recently revealed the SCAR/WAVE
complex as an essential upstream activator of ARP2/3 function in plants. The SCAR/WAVE complex is
conserved from animals to plants and, generally, is composed of the five subunits SCAR/WAVE, PIR121,
NAP125, BRICK and ABI. In plants, four of the five subunits have been shown to participate in trichome and
pavement morphogenesis. Plant ABI-like proteins (ABIL), however, which constitute a small four-member
protein family in Arabidopsis thaliana, have not been characterized functionally, so far. Here we demonstrate
that microRNA knock-down of the ABIL3 gene leads to a distorted trichome phenotype reminiscent of ARP2/3
mutant phenotypes and consistent with a crucial role of the ABIL3 protein in an ARP2/3-activating SCAR/
WAVE complex. In contrast to ARP2/3 mutants, however, the ABIL3 knock-down stimulated cell elongation in
the root, indicating distinct functions of the ABIL3 protein in different tissues. Furthermore, we provide
evidence that ABIL3 associates with microtubules in vivo, opening up the intriguing possibility that ABI-like
proteins have a function in linking SCAR/WAVE-dependent actin nucleation with organization of the
microtubule cytoskeleton.

Keywords: actin, ARP2/3 complex, SCAR/WAVE complex, cytoskeleton, trichomes.

INTRODUCTION
In eukaryotic cells, the polymerization of actin is a funda-
mental process involved in a wide variety of vital cellular
functions including cell polarization, cell motility, membrane
and organelle trafficking, cytokinesis and signaling. There-
fore, actin polymerization has to be temporally and spatially
strictly regulated. Actin monomers have a rather low affinity
for one another and have to form a trimeric seed to initiate
polymerization. This thermodynamically unfavorable reac-
tion can be overcome by accessory proteins facilitating and
accelerating the initiation of actin polymerization, so-called
actin nucleators. The seven-subunit ARP2/3 complex is an
evolutionary highly conserved actin nucleator that has been
found in all eukaryotes including plants (Machesky and
Gould, 1999; Deeks and Hussey, 2005). It initiates the for-
mation of actin branches on the side of preexisting actin
filaments, locally creating a dynamic branched network
of filamentous actin (Blanchoin et al., 2000; Amann and
Pollard, 2001). Loss-of-function mutations in arp2/3 genes
are lethal in most eukaryotes, but not in plants. ARP2/3
mutants have been associated with the distorted group of
trichome mutants of Arabidopsis thaliana (Hulskamp et al.,
1994; Le et al., 2003; Li et al., 2003; Mathur et al., 2003a,b;
El-Din El-Assal et al., 2004; Saedler et al., 2004a; Mathur,
2005). Except for a substantially altered trichome shape,
these mutants are healthy and viable and exhibit only rela-
tively mild phenotypic aberration in cell types other than
trichome cells. This offers a unique opportunity to analyze
the ARP2/3 pathway in a way that is not possible in other
model organisms.
The ARP2/3 complex itself is virtually inactive. To effec-
tively catalyze actin nucleation, it depends on the presence
of further regulatory proteins such as, amongst others, the
Wiskott–Aldrich Syndrome (WASP) proteins or the related
Suppressor of cyclic AMP receptor/WASP family verprolin
homologous (SCAR/WAVE) proteins (Machesky and Insall,
1998; Machesky et al., 1999). While WASP proteins have not
been described in the plant kingdom so far, the existence of
a SCAR/WAVE pathway in plants has been established by a
growing body of evidence accumulated in recent years (for a
review see Szymanski, 2005). In addition to the SCAR/WAVE
proteins, the canonical SCAR/WAVE complex is composed
of four subunits called PIR121 (p53-inducible protein-121,
also named SRA1, CYFIP, GEX2), NAP125 (NCK-associated
protein-125, also known as NAP1, NCKAP1, KETTE, HEM2,

ª 2010 The Authors 925

Journal compilation ª 2010 Blackwell Publishing Ltd

926 Cordula I. Jörgens et al.
GEX3), ABI (Abl-interacting protein) and HSPC300 (also
known as BRICK). The metazoan SCAR/WAVE complex has
recently been shown to be intrinsically inhibited, relying for
its activation upon the simultaneous interaction with acti-
vated small Rho-like GTP binding proteins and acid phos-
pholipids, as well as a specific phosphorylation pattern
(Derivery et al., 2009; Ismail et al., 2009; Lebensohn and
Kirschner, 2009).
In A. thaliana, three of the SCAR/WAVE complex subunits
(PIR121, NAP125 and BRICK) are encoded by single genes
while both, the ABIL proteins and the SCAR/WAVE proteins
are encoded by a small gene family of four genes, respec-
tively. Loss-of-function mutations in the single-copy genes
phenocopy the full range of aberrant phenotypes displayed
by ARP2/3 complex mutants. These include the distorted
trichome phenotype characterized by uncoordinated cell
expansion, less complex pavement cell shape and elongated
petioles and a hypocotyl cell adhesion defect in dark grown
seedlings (Basu et al., 2004; El-Assal Sel et al., 2004; Li et al.,
2004; Saedler et al., 2004b; Zimmermann et al., 2004; Zhang
et al., 2005a; Djakovic et al., 2006; Le et al., 2006). In plants,
therefore, the SCAR/WAVE complex appears to be the major
pathway for the activation of ARP2/3 function. Recently, a
partially redundant role of the SCAR protein family has been
revealed by the characterization of single, double, triple and
quadruple mutants in the SCAR genes (Basu et al., 2005;
Zhang et al., 2005a, 2008; Uhrig et al., 2007). A model for
plant SCAR protein function has been proposed that is based
upon differential quantitative contributions of individual
SCAR proteins to reach a threshold necessary for ARP2/3
complex activation (Zhang et al., 2008).
Furthermore, recent evidence indicates that plant SCAR
proteins might fulfill regulatory functions not uncovered in
other model organisms, so far. Direct protein-protein inter-
actions of SCAR with the activated forms of small Rho-like
GTPases (Rho of plants, ROPs) and with SPIKE1 (SPK1), a
putative DOCK180-type guanine nucleotide exchange factor
(GEF) have been demonstrated. Plant SCAR proteins might
therefore have multiple regulatory roles as effectors of small
GTPases, as part of a signaling complex integrating
upstream cues via contact to a ROP-GEF, and, via its VCA
domain, as direct activator of the ARP2/3 complex (Uhrig
et al., 2007). Integrating genetic data and biochemical
evidence for an association of SPIKE1 with the SCAR/WAVE
complex in vivo, a morphogenetic pathway from SPK1 via
ROP, the SCAR/WAVE and the ARP2/3 complex to the
nucleation of actin has been proposed, recently (Basu et al.,
2008).
The least conserved subunits of the putative SCAR/WAVE
complex in plants are the four predicted ABIL proteins.
Despite very weak sequence homologies, an involvement of
this protein family in SCAR/WAVE complex function has
been proposed based upon the finding that all four ABIL
proteins encoded by the A. thaliana genome are closely
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linked to SCAR proteins, BRICK and NAP125 via direct
protein-protein interactions (Basu et al., 2005; Uhrig et al.,
2007). Furthermore, a physical interaction with SPK1 has
been shown, indicating a potential regulatory function of
ABIL proteins in the context of the proposed morphogenetic
pathway mentioned above (Uhrig et al., 2007; Zhang et al.,
2008). Beyond that, ABIL proteins in plants have not been
functionally characterized, so far.
At present, the role of ABI proteins in the SCAR/Wave
complex has mainly been discussed as being an adaptor
linking the SCAR/WAVE proteins to the NAP125 and PIR121
subunits that in turn are thought to inhibit SCAR/WAVE
function in the absence of an upstream activating signal
(Innocenti et al., 2004; Ismail et al., 2009). However, evi-
dence is accumulating that ABI proteins are involved in a
wide variety of further cellular processes and functions.
These range from direct SCAR/WAVE activating or enhanc-
ing activity to functional roles independent of the SCAR/
WAVE complex, the WASP proteins, or the ARP2/3 complex,
respectively. In mammalian cells the formation of lamelli-
podia and filopodia, respectively require physical interaction
of both, ABI and SCAR/WAVE proteins with the formin
mDia2 (Yang et al., 2007; Beli et al., 2008). Besides its actin-
nucleating activity, mDia2 has been proven to act as a Rho
GTPase effector in formation and orientation of stable
microtubules (Palazzo et al., 2001). Furthermore, ABI-2 has
been shown to act as a tumor suppressor, interacting with
and suppressing the transforming potential of Abelson
kinase, and it plays a role in signaling complexes involving
phosphatidylinositol-3-phosphate kinase (Dai and Pender-
gast, 1995; Shi et al., 1995; Innocenti et al., 2003).
On the whole, the picture of ABI protein function, seems
far from being complete. In this report we provide evidence
that the ABIL3 protein not only is an essential component of
the SCAR/WAVE pathway regulating trichome morphogen-
esis in A. thaliana, but also plays a role in root cell
elongation and provides a molecular link to the microtubule
cytoskeleton.
RESULTS
Molecular analysis of the A. thaliana ABIL gene family
In Arabidopsis thaliana ABIL proteins are encoded by a small
protein family of four members. The four ABIL genes are
predicted to consist of nine exons separated by eight
introns. The predicted ABIL protein sequences are only very
distantly related to ABI proteins from animals or the slime
mold Dictyostelium discoidum, exhibiting only about 15%
sequence conservation restricted to the N-terminal SCAR/
WAVE-binding region of the proteins. Plant ABIL proteins
lack homologies to the homeodomains, proline rich regions
and SH3 domains that are conserved in metazoan ABI pro-
teins. A similarity tree constructed from an alignment of
ABIL protein sequences predicted from seven completely
ª 2010 The Authors

Figure 1. Similarity Tree of ABI and ABI-like Proteins.
An unrooted tree was generated using the Neighbour-joining method (Saitou
and Nei, 1987). Abbreviations used for organisms At, Arabidopsis thaliana;
Mt, Medicago truncatula; Os, Oryza sativa; Pp, Physcomitrella patens; Pt,
Populus trichocarpa; Vv, Vitis vinifera; Zm Zea mays; Dd, Dictyostelium
discoidum; Hs, Homo sapiens; Dm, Drosophila melanogaster. The accession
numbers are listed in Experimental procedures.
sequenced plant genomes reveals two major subfamilies of
plant ABIL proteins (Figure 1). One subclade comprises
proteins similar to the ABIL1 protein from A. thaliana and
includes ABIL proteins from plant genera ranging from
mosses like Physcomitrella patiens to monocots and dicots.
The other subclade, presumably representing a more
derived subfamily, comprises two branches representing
ABIL proteins from monocots and dicots and does not con-
tain a member from the moss Physcomitrella patens. ABIL2
and ABIL3 are closely related and the respective genes are
found in chromosome regions on chromosomes 3 and 5,
respectively, which are products of a recent segmental
genome duplication event.
Expression analysis of A. thaliana ABIL genes
We used quantitative PCR analysis to study the relative
expression levels of the A. thaliana ABIL gene family in
flowers, seedlings, young and old leaves, stems and roots.
While all four ABIL genes are expressed in all tissues
investigated, our data show that there are specific differ-
ences in the expression levels (Figure 2). In seedlings and
stems the expression of all four ABIL genes is uniform and
rather low, while there is higher expression of ABIL2 and
ABIIL4 in flowers and young and older leaves. In roots ABIL1
shows the strongest expression. In all tissues analyzed,
ABIL3 was expressed at comparably low levels. Overlapping
expression domains indicate that, similar to the SCAR
genes, the ABIL genes might act redundantly in A. thaliana
The different levels of expression, on the other hand might –
again similar to the SCAR genes – give rise to different
ª 2010 The Authors
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Function of ABIL3 protein 927
Figure 2. Quantitative analysis of ABIL gene expression in plant organs by
qRT-PCR.
The mRNA levels were normalized to the ACTIN1 gene. Normalized transcript
levels represent the average of two biological samples each measured in
duplicate.
quantitative contributions of individual genes to ABIL
function.
Recently two expression profiling datasets assessing
trichome-specific gene expression in Arabidopsis wild type
and several trichome mutants have ben published (Jakoby
et al., 2008; Marks et al., 2009). Analysis of these data with
respect to expression of ABIL genes revealed that both ABIL1
and ABIL4 expression is decreased in trichomes versus their
expression levels in leaves without trichomes. In contrast,
ABIL3 expression in trichomes is increased as compared to
leaves without trichomes (Figure S1). ABIL2 is not repre-
sented on the Affimetrix chips.
Isolation of ABIL knock-down mutants
To functionally characterize the ABIL gene family in A. tha-
liana, we analyzed publicly available T-DNA insertion lines in
the four ABIL genes. These lines, however, only contain
T-DNA insertions upstream or downstream of the coding
region or in introns. None of these lines exhibited a signifi-
cantly changed mRNA level or altered phenotypes (data not
shown). We therefore used artificial micro RNA (amiRNA) to
knock down gene expression of ABIL genes. An amiRNA
targeting the ABIL3 gene (amiRNA_ABIL3) was expressed in
A. thaliana under the control of the constitutive CaMV 35S
promoter. This construct produced reproducibly a signifi-
cant knock-down of the ABIL3 mRNA and resulted in a visi-
ble aberrant mutant phenotype. We analyzed 38 primary
transformants (T1) and 28 exhibited a mutant trichome
phenotype reminiscent of the distorted phenotypes of
SCAR/WAVE complex mutants (DIS3, GNR, KLK, BRICK) and
ARP2/3 complex mutants (see below). We selected the two
independent lines #15 and #23 for further molecular and
phenotypic analysis. RT-PCR consistently showed that the
ABIL3 mRNA level was significantly decreased (Figure 3).
While generally, the expression of the three other ABIL
genes was unchanged, we observed in one individual plant

928 Cordula I. Jörgens et al.
Figure 3. Transcript Levels of the ABIL genes in wild type and mutant lines.
Transcript levels of the four AtABIL genes were assessed by RT-PCR. Five
individual plants from two independent amiRNA-abil3 lines were compared
to cDNA and genomic DNA from wild type; ACTIN1 was used as a control.
of line #15 (amiRNA-abil3 #15.3) lower expression of the
ABIL1 gene as well, indicating that there might be the
possibility of side-effects on the expression of other ABIL
genes. However, as the mutant phenotype of this plant was
indistinguishable from those of the other plants of line #15
with wild type levels of ABIL1 expression, a significant
contribution of ABIL1 is unlikely. The distorted phenotype
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was consistently found again in the T2 and T3 generations,
demonstrating that the amiRNA-mediated knock-down of
the ABIL3 mRNA was heritable.
To confirm that the amiRNA knock-down of the ABIL3
gene is responsible for the observed mutant phenotype, we
designed ABIL3 and ABIL3-GFP rescue constructs incorpo-
rating silent mutations of essential positions of the predicted
amiRNA binding site in the ABIL3 coding sequence. Both
amiRNA-resistant constructs were indeed able to rescue the
distorted trichome phenotype of amiRNA-abil3 lines #15 and
#23 (Figure 4d,g). In the T3 generation of amiRNA-abil3 lines
#15 and #23 approximately 90% of the plants show the
mutant phenotype, whereas in amiRNA-abil3 lines #15 and
#23 expressing the rescue constructs, two-thirds of the
transformants developed trichomes with a wild type pheno-
type proving the decisive role of ABIL3 in trichome mor-
phogenesis (Figure 4d,g).
Characterization of the ABIL3 amiRNA knock-down
phenotype
The A. thaliana ABIL proteins are predicted to be part of the
SCAR/WAVE complex. Loss-of-function mutations in this
complex, similar to mutations of the ARP2/3 complex result
in the so-called distorted phenotypes affected in the mor-
phogenesis of a number of cell types. We therefore analyzed
the ABIL amiRNA knock-down lines with respect to pheno-
typic aberrations in trichome cells, pavement cell shape,
petiole length, hypocotyl length and root growth character-
istics.
Trichome morphology
Trichomes of the distorted group of mutants are character-
ized by alterations in diameter, in branch length and relative
branch position. To analyze the effect of ABIL3 knock-down
on trichome development we applied the criteria developed
by David Oppenheimer and co-workers (Zhang et al.,
2005b). Both ABIL3 knock-down lines consistently exhibited
irregularly expanded distorted trichomes, and branch
lengths were significantly affected (Figure 4a–c, Table 1).
Figure 4. Phenotype of ABIL3 amiRNA knock-
down lines.
The effect of ABIL3 knock-down on trichome cell
morphology was analyzed by scanning electron
microscopy. Wild type (a, e) is compared to two
individual lines of amiRNA lines [line #15 (B), and
line #23 (c, f)]. The mutant trichome phenotype of
the amiRNA knock-down line #23 was rescued by
constitutive expression of an amiRNA-resistant
ABIL3 construct (d, g). Scale bars: 75 lm (a–d),
500 lm (e–g).
ª 2010 The Authors
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Function of ABIL3 protein 929

Table 1 Quantitative analysis of the trichome phenotype of wild above the first branching point, respectively. Furthermore,
type and amiRNA-ABIL3 lines the mutant trichomes exhibited a significantly increased
amiRNA-ABIL3 maximum diameter at the base (Table 1). Application of
Student’s t-test to our measurements confirmed the statisti-
Wild type (Col0) #15.1 #23.3 cal significance of the differences in interbranch zone lengths
Trichome branch length (lm) (P = 1.44*10)11 and P = 1.09*10)11, respectively) and tri-
Branch 1 183  36.2 43  24.8a 53  27.5a chome diameter (P = 0.075 and P = 0.029, respectively)
Branch 2 175  53.3 72  41.7a 95  44.1a between wild type and ABIL3 amiRNA knock-down mutants.
Branch 3 133  39.4 53  31.1a 67  51.7a
Interbranch zone 33  6.8 74  25.6a 70  21.1a Actin cytoskeleton in trichomes
length (lm)
Maximum 32  6.1 40  7.9a 36  6.7a Stable expression of the actin-binding domain of fimbrin
diameter (lm) fused to GFP in wild type and ABIL3 amiRNA knock down
lines revealed specific differences in the organization of the
n = 30. actin cytoskeleton in developing trichome cells. While wild
Mean value  SD. type trichomes in stage 3/4 (development and expansion of
a
Significantly different from wild type.
branches, Szymanski et al., 1999) exhibit actin filaments that
loosely align with the long axis of the developing branch, the
Analysis of 10 trichome cells on three leaves of equal actin filaments in the mutants are generally more bundled
position from each Abi amiRNA line revealed that in the and more randomly distributed (Figure 5). Similar aberra-
mutants all three branches were significantly shorter than tions in the organization of the actin cytoskeleton have been
the respective branches of wild type trichomes. The first described earlier for other SCAR/WAVE complex loss-of-
branch was reduced in length to an extent similar to function mutants (Basu et al., 2004, 2005; Brembu et al.,
those found in the full distorted phenotypes of ARP2/3 2004; El-Assal Sel et al., 2004; Li et al., 2004; Dyachok et al.,
mutants growing to only about one-fourth of the length of 2008).
wild type branch one. Branches two and three however,
Pavement cell shape
while still significantly reduced in length, are somewhat
less affected as compared to other distorted mutants In A. thaliana, activity of the ARP2/3 complex and likewise
(Figure 4a–c, Table 1). the SCAR/WAVE complex is required for cell shape devel-
The branching point positions relative to the trichome cell opment of epidermal pavement cells. In strong distorted
axis are affected in previously characterized ARP2/3 and mutants, lobing of pavement cells is strongly reduced. We
SCAR/WAVE mutants. Therefore, we analyzed the distance quantified the lobe formation by calculating the complexity
between first and second branch point and the diameter of of 99 individual pavement cells within the apical third of ten
the stem at the basis of the trichome cells in the ABIL3 mature rosette leaves per mutant line using the formula:
amiRNA knock-down lines. The interbranch zone between complexity ¼ ðperimeterÞ2 =ð4  p  areaÞ. While the two ABIL3
branches one and two was statistically significantly pro- amiRNA lines exhibit the distorted trichome phenotype to
longed by a factor of more than two (Table 1). While in wild almost full extent, the complexity of pavement cell shape
type trichomes the distance between the first and the second was only slightly affected in these mutants, with a statisti-
branch was on average 33 lm, in the two ABIL3 amiRNA cally significant alteration only in the case of amiRNA line
lines, the second branching point was found 74 and 70 lm #23 (Figure S2, Table S1).

Figure 5. Actin cytoskeleton in ABIL3 amiRNA

knock-down lines.
The actin cytoskeleton was visualized by stably
expressing GFP-ABD2-GFP in wildtype Arabid-
opsis (a) and an ABIL3 amiRNA knock-down line
(b). ABIL3 knock-down leads to increased bun-
dling of actin filaments in trichome cells. Scale
bars: 50 lm.

ª 2010 The Authors

Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 925–935
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930 Cordula I. Jörgens et al.

In ARP2/3 complex mutations and mutations of ARP2/3

complex regulators, gaps between epidermal cotyledon
cells indicate that ARP2/3 function is involved in cell–cell
adhesion following intercalation of lobes of adjacent pave-
ment cells (Basu et al., 2004). The two ABIL3 amiRNA knock-
down lines investigated in this study did not exhibit any
gaps between epidermal pavement cells and therefore,
apparently ABIL3 is not involved in this function of the
SCAR/WAVE complex or masked by redundancy with other
ABIL genes.

Hypocotyls, petioles and root growth characteristics

In knock-out mutants of ARP2/3 complex subunits and like-
wise SCAR/WAVE complex mutants, hypocotyl elongation is
substantially inhibited, while petiole elongation is enhanced
in the dark (Li et al., 2004). Furthermore, in ARP2/3 complex
mutants and scar quadruple mutants as well as brick knock-
out mutants root growth rate and the ability to penetrate
semi-solid media is strongly impaired (Dyachok et al., 2008).
In contrast, knock-out mutants of the PIR121 and NAP124
subunits of the SCAR/WAVE complex, respectively, exhib-
ited significantly increased root lengths (Li et al., 2004).
Both ABIL3 amiRNA lines did not exhibit any significant
reduction in hypocotyl elongation (Table 2) or a significant
increase in cotyledon petiole length (data not shown). Root
length was measured in light-grown A. thaliana Col0 wild
type and in the two ABIL3 amiRNA lines between 3 and
8.5 days after germination (Figure 6). In comparison to wild
type, the two ABIL3 knock-down lines showed a significant
increase in root length (Figure 6, t-test P = 1.3*10)3 and
3.9*10)6 for lines #15 and #23, respectively). In dark-grown
seedlings, 17 days after germination, this root elongation
difference between wild type and the ABIL3 amiRNA knock-
down lines was even more obvious (Table 2). Root length in
the mutants was increased by a factor of approximately 1.4
as compared to wild type. Similarly, knock out mutants of
pir121 and nap125 exhibited a comparable increase in root
length (Table 2). The observed increased root length of the
ABIL and pir121 mutants was mainly based on an increased
cell elongation (Table 2), although in the case of the ABIL3
Figure 6. Effect of ABIL3 knock-down on root elongation.
Root length of two individual lines expressing amiRNA-abil3 is compared to
wild type and scar2/4 mutant plants over a period of 8 days grown on vertical
agar plates. Error bars represent standard deviation.
mutants, cell proliferation might play an additional role,
because the measured increase in cell length does not quite
match the relative increase in root length. Root rigidity, as
measured by an agar penetration assay, was not altered in
the ABIL3 amiRNA mutants compared to the wild type
(Table 2).
ABIL3 associates with microtubules in plant cells
Stable expression of a C-terminal fusion of the ABIL3 protein
with GFP rescued the amiRNA-induced aberrant trichome
phenotype (see above), indicating that this construct is
functional in vivo. However, protein levels of this constructs
were apparently very low, as we were not able to detect GFP
fluorescence in the rescued lines. To analyze the intracellular
localization of ABIL3 we therefore transiently expressed the
C-terminally GFP-tagged ABIL3 protein in tobacco leaves.
Surprisingly, ABIL3 associated with filamentous structures
reminiscent of cortical microtubules (Figure 7a). Applying
the actin disrupting drug Latrunculin B did not affect the
filamentous structures labelled by ABIL3-GFP (Figure 7b).
On the other hand, incubation of the leaves in the micro-
tubule-destabilizing drug Oryzalin disrupted the filamentous

Table 2 Comparison of root growth and hypocotyl growth of wild type, amiRNA-ABIL3 lines and scar2/4 mutants

Root length 17 Cell length in root Agar penetration Hypocotyl length

DAG (mm) elongation zone (lm) 1.25% Agar n > 30 (cm) n = 31

Wild type (Col0) 14.4  3.8 (n = 28) 87.5  21.5 (n = 95) 95% 2.12  0.24
amiRNA-ABIL3 #15 20.0  4.7a (n = 24) 102.4  32.3a (n = 117) 97% 2.23  0.29
amiRNA-ABIL #23 20.1  4.8a (n = 24) 100.2  20.5a (n = 96) 95% 2.26  0.19
nap125 18.0  4.2a na na na
pir121 20.1  3.8a 136.5  24.9a (n = 14) na na

Mean value  SD.

a
Significantly different from wild type.
na, Not analysed.

ª 2010 The Authors

Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 925–935

Figure 7. ABIL3-GFP associates with micro-
tubules.
Transiently expressed ABIL3-GFP associates
with filamentous structures in N. benthamiana
leaves (a). Treatment with the actin depolymer-
izing drug Latrunculin B (50 lM, 48 h) did not
affect the filamentous structures labeled with
ABIL3-GFP (b), while treatment with the micro-
tubule-destabilizing drug Oryzalin (20 lM, 48 h)
disrupts ABIL3-labeled filaments. Co-expression
of ABIL3-GFP (d) with microtubule-associated
MAP64-7-RFP (e) resulted in co-localization (f,
overlay of d and e) in filamentous structures that
are sensitive to Oryzalin treatment (g). Scale
bars: 50 lm).
structures providing evidence, that in plant cells ABIL3
associates with microtubules (Figure 7c). Furthermore,
ABIL3-GFP co-localizes with the microtubule-binding protein
MAP65-7-RFP at filamentous structures that are sensitive to
Oryzalin treatment (Figure 7d–f).
DISCUSSION
Recent advances in the molecular characterization of the
DISTORTED group of A. thaliana trichome mutants have
established an ARP2/3-dependent actin nucleation pathway
underlying cell shape development of epidermal cells in
plants (Mathur, 2005). A pentameric SCAR/WAVE complex
has been proposed as the major upstream activator regu-
lating ARP2/3 function in A. thaliana (for reviews see Deeks
and Hussey, 2005; Szymanski, 2005). Experimental proof,
however, has been provided for only four of the predicted
five subunits of plant SCAR/WAVE complexes, so far (Basu
et al., 2004, 2005; El-Assal Sel et al., 2004; Li et al., 2004;
Saedler et al., 2004b; Zimmermann et al., 2004; Zhang et al.,
2005a; Djakovic et al., 2006; Uhrig et al., 2007). In this study
we provide functional evidence for an essential role of the
ABIL3 protein in ARP2/3-dependent trichome morphogene-
sis in A. thaliana.
Knock-down of ABIL3 by an amiRNA approach results in
an aberrant organization of the actin cytoskeleton and a
trichome phenotype reminiscent of the distorted group of
mutants. These mutants are characterized by an uncoordi-
nated expansion of trichome cells and exhibit additional
defects in cell morphogenesis of leaf pavement cells and in
hypocotyl cell elongation and adherence (Hulskamp et al.,
1994; Le et al., 2003; Mathur et al., 2003a,b; Basu et al., 2004;
Brembu et al., 2004; Deeks et al., 2004; El-Din El-Assal et al.,
2004; Saedler et al., 2004a) While strong ABIL3 amiRNA
knock-down mutants exhibit the distorted trichome pheno-
type to almost full extent, other tissues and cell types are
only weakly or not affected at all. We observed a slight
reduction in pavement cell complexity indicating a weak
ª 2010 The Authors
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Function of ABIL3 protein 931
mutant lobing phenotype, but no reduction in hypocotyl
length and no cell–cell adhesion defects. In trichomes,
therefore, amongst the four ABIL paralogs encoded by the
A. thaliana genome, ABIL3 appears to be quantitatively the
major component necessary for proper cell morphogenesis.
This conclusion is supported by the fact that the mutant
phenotype can be rescued by expression of an amiRNA-
resistant ABIL3 gene in the amiRNA knock-down lines.
We have previously shown that the ABIL3 protein inter-
acts with the SCAR, BRICK and NAP125 subunits of the
SCAR/WAVE complex (Uhrig et al., 2007). The finding that
the ABIL3 amiRNA knock-down lines share the distorted
trichome phenotype and the aberrant bundling and arrange-
ment of the actin cytoskeleton with scar/wave, nap125, brick
and pir121 mutants supports the conclusion that the A. tha-
liana ABIL proteins, similar to their metazoan homologs,
have an essential function in the context of a pentameric
SCAR/WAVE complex. The four ABIL genes and four genu-
ine SCAR genes in the A. thaliana genome allow the
formation of sixteen different complexes. Together with
the recent findings that the SCAR2 protein accounts for most
of the SCAR/WAVE complex function in trichomes, and that
SCAR2 and ABIL3 physically interact (Basu et al., 2005;
Zhang et al., 2005a, 2008; Uhrig et al., 2007), it is likely that
the particular complex formed by PIR121, NAP125, BRICK,
ABIL3 and SCAR2 is the main regulator of trichome cell
morphogenesis in A. thaliana.
Recent surveys of the expression profile of trichome cells
revealed that both ABIL1 and ABIL4 are expressed in
trichome cells, although somewhat lower than ABIL3
(Jakoby et al., 2008). An interesting question is therefore,
whether in trichomes ABIL3 protein function is different
from ABIL1 and ABIL4 in a way that these two ABIL proteins
cannot compensate for a loss of ABIL3, or whether the
proteins are functionally redundant and the knock-down of
ABIL3 leads to total ABIL protein levels below a critical
threshold level necessary for ARP2/3 activation. This would

932 Cordula I. Jörgens et al.
be in line with the threshold model proposed recently for
SCAR/WAVE protein function in A. thaliana (Zhang et al.,
2008).
The ABIL proteins are, in terms of sequence homology,
the least conserved subunit of the plant SCAR/WAVE
complex. All metazoan ABI proteins characterized so far
are composed of four conserved domains: an N-terminal WA
domain, a domain with similarity to homeodomains, a
proline-rich domain and a C-terminal SH3 domain (Innocenti
et al., 2004). Plant ABIL proteins are different. As predicted
from genome sequences, they exhibit limited sequence
homologies to the WA domains but completely lack any
protein domains similar to the other three domains con-
served in the canonical ABI protein family. The WA domain
is necessary for the interaction of metazoan ABI proteins
with SCAR/WAVE proteins and the BRICK protein (Innocenti
et al., 2004). These interactions are conserved in plants
where ABIL proteins are able to interact with both, SCAR/
WAVE and BRICK proteins (Basu et al., 2005; Uhrig et al.,
2007). The data presented in this study show that plant ABIL
proteins are presumably acting in a functional SCAR/WAVE
complex and therefore suggest that the other three con-
served domains missing in plant ABIL proteins are not
necessary for SCAR/WAVE complex assembly and activity.
More likely they might have functions in metazoan-specific
regulatory processes. This notion fits with the recent finding
that a truncated mammalian ABI-2 protein lacking the
conserved C-terminal proline-rich region and the SH3
domain, readily incorporates in a reconstituted pentameric
SCAR/WAVE complex, which similar to its native counter-
part is intrinsically inhibited (Ismail et al., 2009).
There might be, however, further functions of ABIL
proteins in plants that do not fit a simple adapter function
connecting the SCAR/WAVE proteins with the inhibitory
subunits of the complex. SCAR/WAVE and BRICK mutants
have recently been found to exhibit significantly decreased
root elongation (Djakovic et al., 2006; Zhang et al., 2008). We
found that the knock-down of the ABIL3 gene, and similarly
the knock-out of pir121 and nap125 lead to a significant
increase in root length that was due to an increased cell
length in the elongation zone. Knock-out of PIR121 or
NAP125, respectively, has previously been reported to result
in an even more pronounced effect on root elongation
leading to roots more than twice as long as the wild type
roots (Li et al., 2004).
This contrasting effect of ABIL, PIR121 and NAP125 knock-
outs on the one hand and SCAR/WAVE, BRICK and ARP2/3
knock-outs on the other hand could indicate that inhibition
of root elongation might be a function of PIR121, NAP125
and ABIL proteins that is independent of the SCAR or BRICK
proteins. Alternatively, loss-of-function mutations removing
PIR121, NAP125 or ABIL function in the roots, respectively,
might lead to a de-repression of the root growth-promoting
activity of SCAR/WAVE proteins. With regard to trichome
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and pavement morphogenesis, genetic evidence from plant
SCAR/WAVE complex mutants are consistent with the idea
that the whole complex is necessary for activity. This is in
line with the activator model of SCAR/WAVE complex
function, postulating an inactive pentameric complex that
is activated as a whole by upstream signals. However, the
surprising root phenotypes of pir121, nap125 and ABIL3
mutants, interestingly, would fit with an over-activation of
SCAR function, in line with the dissociation model that relies
upon the de-repression of SCAR/WAVE activity by a signal-
induced dissociation of a SCAR-BRICK dimer from the
pentameric complex. The dissociation model has been
proposed on the basis of in vitro reconstituted SCAR/WAVE
complexes (Eden et al., 2002). More recent biochemical data
however, show that a complex composed of human WAVE2,
ABI-1, NAP1 and PIR121 is active as a whole, challenging the
‘dissociation model’ and supporting the ‘activation model’
(Innocenti et al., 2004). Furthermore, very recently it was
shown that reconstituted recombinant human and Drosoph-
ila SCAR/WAVE complexes are inactive and can be activated
without disassembling by coincident signals including
interaction with GTP-bound Rac proteins, interaction with
acid phospholipids and a specific state of phosphorylation
(Ismail et al., 2009; Lebensohn and Kirschner, 2009). How-
ever, there are a number of studies providing genetic
evidence that corroborates the dissociation model in vivo
(Blagg et al., 2003; Bogdan and Klambt, 2003; Pollitt and
Insall, 2008). A. thaliana and the conflicting root elongation
phenotypes of scar/wave and brick mutants on the one hand,
and pir121, nap125 and abiL3 mutants on the other hand
might emerge as a promising model system to help
dissecting the molecular mechanisms of SCAR/WAVE com-
plex function.
Many aspects of cell growth and development require a
coordination of dyamic microtubules and actin microfila-
ments. While in animals numerous studies have addressed
this question, in plants, molecular insight in microtubule-
microfilament cross-talk is only emerging (reviewed in
Petrasek and Schwarzerova, 2009). Cross-regulation
between actin and microtubule organization underlies the
morphogenesis of the interdigitating pavement cells in
A. thaliana. Here, the small GTPases ROP2 and ROP6
together with their effector proteins RIC1 and RIC4 have
counteracting functions on cytoskeleton dynamics: ROP2
promotes the formation of fine actin patches in the lobe
regions and simultaneously inhibits microtubule alignment,
while ROP6 inhibits actin patch formation and stimulates
microtubule alignment in the neck regions (Fu et al., 2005,
2009). In trichome development a correlative connection
between these two cytoskeleton elements has been
observed in some of the ARP2/3 complex loss-of-function
mutants, which in addition to the actin aberrations exhibited
altered microtubule organization as well (Schwab et al.,
2003; Saedler et al., 2004a). The molecular basis underlying
ª 2010 The Authors

these observations is not understood, so far. Our intriguing
finding that the ABIL3 protein associates with microtubules
will pave the way for dissecting actin-microtubule cross-talk
in molecular detail. Once more, the fact that SCAR/WAVE
and ARP2/3 genes are not essential genes in Arabidopsis
thaliana will allow detailed mutational analyses that should
make it possible to separate the microtubule-binding func-
tion from the SCAR/WAVE-dependent function of ABIL
proteins and thereby will be instrumental in understanding
the complex network underlying cell morphogenesis in
plants.
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Arabidopsis thaliana seeds were sterilized with 75% ethanol fol-
lowed by several washings with sterile water and stratified at 4C for
2–3 days. Plants were grown on 1·MS media with 1% sucrose and
1% phytoagar or in soil under long day conditions at 20C unless
otherwise stated. To analyze the etiolated hypocotyl and petiole
phenotypes, plants were grown on 1/2MS with 1% sucrose in the
dark after 7 h of light induction.
Constructs and recombinant DNA manipulations
Standard molecular biological technologies such as growth of
bacteria, plasmid isolation, and PCR were performed as described
by Sambrook et al. (1989).
The amiRNA sequence was designed with the help of WMD2
WebMicroRNA Designer (http://www.weigelworld.org). For subcl-
oning, the miRNA 319 precursor was amplified from genomic Col0
DNA using Primer P1 (5¢-CAAACACACGCTCGGACGCATATTACAC-
3¢) and P2 (5¢-CCATGGCGATGCCTTAAATAAAGATAAACC-3¢) con-
taining attB1 and attB2 sites for GATEWAY recombination (Invitro-
gen, http://www.invitrogen.com). The PCR product was recombined
into pDONR201 (Invitrogen) to yield pENTR319. To replace the
endogenous miRNA and miRNA* with the artificial miRNA
sequence of ABIL3 (5¢-TTAGTTCGAAATACTCGGTTG-3¢), Eco31I
restriction sites were engineered into the miRNA319 containing
plasmid, using inverse PCR with oligos P3 (5¢-GAGA-
GGTCTCGTCTACATATATATTCCTAAAACATCAATTC-3¢) and P4
(5¢-GAGAGGTCTCCTCTCTCTTTTGTATTCCAATTTTCTTGATTAATC-3¢).
The amiRNA of ABIL3 was constructed using a PCR approach
with the primers P5 (5¢-GAGAGGTCTCAGAGACAACCGAGTAT-
TTCGAACTAATCAAAGAGAATCAATGATCCAATTTGTC-3¢) and P6
(5¢-GAGAGGTCTCGTAGACACCCGAGTATTTGGAACTATTCACAGG-
TCGTGATATGATTCAATTAGCTTC-3¢) followed by restriction and
ligation into pENTR319. The resulting amiRNA loop structure,
containing the ABIL-specific sequence, was transferred in the plant
transformation vector pBatTL-B-GFP under the control of the
cauliflower mosaic virus 35S promoter, resulting in pBatTL-B-GFP-
amiRNA_ABIL3. The 35s::amiRNA_ ABIL construct was transformed
into wild type A. thaliana Col0 plants.
In the ABIL3 rescue construct silent mutations of the predicted
amiRNA binding site of the ABIL3 coding sequence were incorpo-
rated by overlapping PCR using oligos P7 (5¢-AT-
GAGTGCAGCAGCTACAATGCC-3¢) and P8 (5¢-AGGGTCATTACTG
ATCGACAGCTGCTGAATACAACTGTGTT-3¢); and P9 (5¢-CAG-
CTGTCGATCAGTAATTACCCTACACAAACGATGAACAAAA-3¢) and
P10 (5¢-TTAATATTCGTCCAAATAAGTG-3¢).The two PCR products
are subsequently fused in a third PCR using Primers P7 and P10. The
rescue construct was transferred in the plant transformation vector
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 925–935
1365313x, 2010, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2010.04210.x by Cochrane Germany, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Function of ABIL3 protein 933
pBatTL-K-GFP under the control of the cauliflower mosaic virus 35S
promoter. The ABIL3 rescue construct was transformed into two
ABIL3 amiRNA lines and wild type A. thaliana Col0 plants.
Agro-infiltration and analysis of intracellular localization
To visualize actin cytoskeleton in trichomes, intact GFP:ABD2:GFP
(Wang et al., 2008) expressing 10-14 days old seedlings were used.
F-Actin was visualized using a Leica SP2 confocal laser scanning
microscope (http://www.leica.com).
For localization studies of ABIL3 the full length cDNA was
subcloned into pBatTL-B-GFP (JFU, unpublished) to form a
C-terminal fusion with GFP. Functionality of the ABIL3-GFP fusion
protein was proven by rescue of the two ABIL3 amiRNA knock-down
lines. The construct was infiltrated into Nicotiana benthamiana
plants. Agrobacterium tumefaciens strain LBA4404 was grown to
mid exponential phase, centrifuged and resuspended to an OD600
of 0.8 with the infiltration medium (10 mM MES, pH 5.6, 10 mM
MgCl2, 200 mM acetosyringone). For co-infiltrations, equal volumes
of cultures were mixed before agro-infiltration. GFP fluorescence
was detectable 3–5 days after infiltration. Oryzalin and Latrunculin B
were prepared as 10 mM stocks in DMSO and 100% Ethanol,
respectively. The drugs were infiltrated 3 days after agro-infiltration
of the leaves. Intracellular localization was analyzed using Leica SP2
confocal laser scanning microscope.
Plant morphology
Pavement cell shape was analysed using agarose imprints of rosette
leaves (Mathur and Koncz, 1997). To measure the trichome param-
eters, hypocotyl length and to determine the complexity of pave-
ment cells, images were acquired using a Leica DMRB microscope
equipped with a high resolution KY-F70-B camera (JVC, http://
jdl.jvc-europe.com) and DISKUS software (DISKUS, http://
www.hilgers.com).
For scanning electron micrographs freshly cut leaves were
mounted without fixing and immediately scanned with a scanning
electron microscope (Leo Electron Microscopy, http://www.smt.
zeiss.com/leo).
Analysis of gene expression
Total RNA was isolated by using TRI reagent (Sigma, http://www.
sigmaaldrich.com) from the following tissues of wild type A. thali-
ana plants: seedlings, roots, young and old rosette leaves, stems
and flowers. For the gene expression analysis of the amiRNA lines
4 week old leaves were used for mRNA isolation. The RNA was
treated with DNaseI (Fermentas, http://fermentas.com). First strand
cDNA was then synthesized with the RevertAid H-Minus First Strand
cDNA Synthesis Kit (MBI). The following primers were used for
tissue expression analysis: abil1-fw (5¢-TGCATCGAAATCACTCT-
CCTGGC-3¢), abil1-rev (5¢-TGAATCTTTGGAGCTTGGTGCAACT-3¢);
abil2-fw (5¢-ACCAACGACACCAAGCAAGAGC-3¢), abil2-rev (5¢-AAT-
GCGACTCGGACCGAAGC-3¢); abil3-fw (5¢-GTTCCCAGCAATCACTT-
GTGATCG-3¢), abil3-rev (5¢-AGCTTTGCGAGGTTGCCACC-3¢); abil4-
fw (5¢-AACAACATCAACAACAGAACACCAA-3¢), abil4-rev (5¢-CCTG-
AACGTAAGAGAGGGAATCTCA-3¢); ACTIN1fw (5¢-TGCGACAATG-
GAACTGGAATG-3¢), ACTIN1rev (5¢-GATAGCATGTGGAAGTGCA-
TAC-3¢).
Expression of ABIL genes in the amRNA lines was detected using
the following primers: abil1-fw, abil1-rev2 (5¢-CGAGACATAGCCA-
GCTTTGAG-3¢); abil2-fw2 (5¢- GCGGGTGAAATTATGACTGCTAC-3¢),
abil2-rev; abil3-fw (5¢-ATGAGTGCAGCAGCTACAATGCC-3¢), abil3-
rev; abil4-fw, abil4-rev2 (5¢-TTAGTTTCTCGACTTGCTCATACTC-3¢).
Real-time RT-PCR was performed with the GeneAmp 5700
sequence detection system (Applied Biosystems, http://www3.

934 Cordula I. Jörgens et al.
appliedbiosystems.com) using SYBR Green PCR master mix kit
(Applied Biosystems) according to the manufacturer’s instructions.
Transcript levels of ABIL1, ABIL2, ABIL3 and ABIL4 genes in the RNA
samples were normalized with transcript level of A. thaliana Actin1
to allow quantification of gene expression relative to an endoge-
nous control. For each sample duplicate reactions of two biological
replicates were performed. Relative quantification of expression
levels was performed by using the equation for normalized expres-
sion [DCt (cycles to threshold) = CT (ABIL genes) ) CT (ACTIN1 gene);
normalized expression level = (2DCT )].
Homology searches were performed using BLAST programs
(Altschul et al., 1990), and amino acid alignments were performed
using ClustalW multiple sequence alignment-based programs
(http://ebi.ac.uk/Tools/clustalW2/).
Accession numbers
ABIL1 (AT2G46225), ABIL2 (AT3G49290.1), ABIL3 (At5g24310),
ABIL4 (AT5G42030.1), ATSCAR2 (AT2G38440), AtSCAR4
(AT5G01730); OsABIL1 (NP_001043613), OsABIL2 (NP_001043613),
OsABIL3 (NP_001042529.1), OsABIL4 (EAZ12300), OsABIL5
(NP_001043308.1); ZmABIL1 (ACG3497), ZmABIL2 (ACG39415),
ZmABIL3 (ACG32122); PpABIL1 (EDQ51849), PpABIL2(EDQ68226);
PtABIL1 (EEE80657), PtABIL2 (EEE98482), PtABIL3 (EEE98482.1),
PtABIL4 (EEE84256), PtABIL5 (EEE78648); VvABIL1 (XP_002279419),
VvABIL2 (XP_002264080), VvABIL3 (XP_002277097); MtABIL1
(AC182579_28.3), MtABiL2 (CR956619_5.4), MtABIL3 (AC149127_
2.3), MtABIL4 (AC134823_13.4); DdAbi (XP_647444.1), DmAbi
(NP_477263); HsABI1 (NP_005461.2).
ACKNOWLEDGEMENTS
The work was supported by grants from the Deutsche Fors-
chungsgemeinschaft (DFG) and the International Graduate School
in Genetics and Functional Genomics (IGS-GFG) at the University of
Cologne. We thank Ellison Blancaflor for providing the actin marker
GFP-ABD2 for this study.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Expression levels of Arabidopsis ABIL genes.
Figure S2. Leaf epidermal cells of ABIL3 amiRNA knock-down lines.
Table S1. Quantitative analysis of the complexity of pavement cells
of wild type and amiRNA-ABIL3 lines.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
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