Plant Cell Physiol. 45(6): 734–741 (2004)
JSPP © 2004
The enl Mutants Enhance the lrx1 Root Hair Mutant Phenotype of
Arabidopsis thaliana
Anouck Diet 1, Susanne Brunner 1 and Christoph Ringli 2
Institute of Plant Biology, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland
;
small region at the tip through the fusion of vesicles containing
new cell wall material with the plasma membrane (Cai et al.
1997).
Root hair development is a process that has received considerable attention in the last years and several mutants affected
in cell fate determination and root hair formation have been
isolated. wer, ttg, gl2, erh3, and rhd6 mutants are affected in
the cell specification process and are characterized by either
ectopic or strongly reduced root hair formation (for review see
Schiefelbein 2000, Webb et al. 2002). The position of root hair
emergence on the epidermal cell is influenced by several factors. rhd6, trh1 and der1 are three mutants that frequently show
positioning of the root hair away from the distal end of the cell
(Masucci and Schiefelbein 1994, Rigas et al. 2001, Ringli et al.
2002). A number of Arabidopsis mutants in root hair development have been identified. Their phenotypes include the formation of root hairs that are short, enlarged, wavy, form spherical
structures at the root hair base or frequently collapse (for
review, see Grierson et al. 2001). Some of these mutants were
cloned and revealed a plethora of cellular machineries that are
involved in root hair formation. K+ transport across the plasma
membrane (Rigas et al. 2001) as well as a Ca2+ gradient in the
region of growth (Foreman et al. 2003) are important for tip
growth. Intracellular components such as a GTP-binding protein (Wang et al. 1997) and a protein kinase (Oyama et al.
2002) have also been identified. Mutations in a cellulose synthase-like protein and in a UDP-galactose-epimerase cause a
weakening of the extracellular matrix and lead to swollen and
frequently burst root hair structures (Favery et al. 2001, Seifert
et al. 2002). The actin cytoskeleton is important for cell polarity and plays a major role in root hair growth. Actin monomers
polymerize to form actin filaments and the application of actin
filament-interfering compounds leads to aberrant root hair initiation and the stop of tip growth (Holmes et al. 1990, Braun et
al. 1999, Miller et al. 1999). The map-based cloning of der1, a
root hair mutant affected in actin2, provided the genetic proof
that the actin cytoskeleton is indeed essential for root hair
development (Ringli et al. 2002).
We have characterized LRX1, a chimeric extracellular protein consisting of an LRR domain and a C-terminal extensin
domain. LRX1 is specifically expressed in root hairs and lrx1
mutants develop a strong mutant phenotype with root hairs that
are short, frequently burst and often form bulbous structures at
The development of root hairs serves as an excellent
model to study cell growth using both cytological and
genetic approaches. In the past, we have characterized
LRX1, an extracellular protein of Arabidopsis consisting of
an LRR-domain and a structural extensin domain. LRX1 is
specifically expressed in root hairs and lrx1 mutants show
severe deficiencies in root hair development. In this work,
we describe the characterization of enl (enhancer of lrx1)
mutants that were isolated in a visual screen of an ethylmethanesulfonate -mutagenized lrx1 line for plants exhibiting an enhanced lrx1 phenotype. Four recessive enl mutants
were analyzed, three of which define new genetic loci
involved in root hair development. The mutations at the enl
loci and lrx1 result in additive phenotypes in enl/lrx1 double
mutants. One enl mutant is affected in the ACTIN2 gene
and encodes a protein with a 22 amino acid deletion at the
C-terminus. The comparison of molecular and phenotypic
data of different actin2 alleles suggests that the truncated
ACTIN2 protein is still partially functional.
Keywords: ACTIN2 — Arabidopsis — enl — lrx1 — Root
hair.
Abbreviations: enl, enhancer of lrx1; CAPS, cleaved amplified
polymorphic sequence; DICM, differential interference contrast microscopy; EMS, ethylmethanesulfonate.
Introduction
Root hair cells are specialized root epidermal cells with
long thin extensions that protrude perpendicularly from the root
surface. Because root hairs are formed by a single cell and are
easily accessible for analysis, they provide an excellent system
to study cell growth and development. Epidermal cells differentiate into root hair cells (trichoblasts) upon positional cues relative to the underlying cortical cells (Dolan et al. 1994). Root
hair development starts with an initial bulge formation at the
distal end of the cell. Subsequently, a tip-growth machinery initiates slow tip growth on the bulge. In the last phase, tip growth
becomes a fast process, resulting in a long root hair proper
(Dolan et al. 1994). Tip growth is an extreme form of polarized growth in which cell expansion takes place only in a very
1
2
These authors contributed equally to this work.
Corresponding author: E-mail, [email protected]; Fax, +41-1-634-82-04.
734
enl mutants enhance the lrx1 mutant phenotype
735
Fig. 1 Mutant phenotypes of the enl/lrx1 double mutants. Seedlings were grown for 4 d. Only the roots of the seedlings are shown. (A) Overview on the root hair phenotypes. (B) Enlargement of the root hairs. Col, wild-type Columbia; lrx1, lrx1-1s allele used for the EMS mutagenesis.
(A) Bar = 500 µm; (B) Bar = 250 µm.
the root hair base (Baumberger et al. 2001). Analysis of LRX2,
a paralog of LRX1 (Baumberger et al. 2003a), has revealed that
LRX1 and LRX2 synergistically interact during root hair formation. While an lrx2 knock-out mutant does not show an aberrant phenotype, the lrx1/lrx2 double mutant is strongly affected
in root hair development, and barely develops root hairs.
Electron-microscopic analysis points towards a function of
LRX1 and LRX2 in the development of the extracellular
matrix, as lrx1/lrx2 double mutants show major alterations in
the structure of the cell wall (Baumberger et al. 2003b).
Here, we describe the isolation of enl (enhancer of lrx1)
mutants that were isolated by screening an ethylmethanesulfonate (EMS)-mutagenized lrx1 population for plants displaying a stronger lrx1 phenotype. Four enl lines with different phenotypic characteristics were analyzed. Comparison of the enl/
lrx1 double mutant phenotypes with the corresponding enl single mutants revealed that the enl mutations give rise to lrx1independent phenotypes and that they cause additive effects
with lrx1 on root hair development. The genetic mapping of the
mutations and complementation experiments showed that enl1,
enl5, and enl7 are new, so far unidentified root hair mutants,
whereas enl2 is an actin2 allele. Interestingly, the comparison
of the enl2 mutation with the previously isolated actin2 mutant
alleles of der1 (Ringli et al. 2002) suggests that the enl2 mutation, which causes a C-terminal truncation of ACTIN2 by 22
amino acids, still results in a partially functional protein.
Results
Isolation of enl mutants
An lrx1 line was identified in which the En-1 transposon
located in the LRR-coding region of the originally isolated
lrx1-1 allele (Baumberger et al. 2001) had excised, leaving a
6-bp deletion. The phenotype of this stable lrx1-1 allele
(referred to as lrx1-1s) was phenotypically indistinguishable
from lrx1-1, indicating that the 6-bp deletion has a strong
impact on the activity of LRX1. The lrx1-1s allele was used for
EMS mutagenesis. Mutagenized M1 plants were grown in 200
pools of 10 plants each and propagated to the next generation.
Two hundred M2 seedlings per pool were grown for 4 d on
plates in a vertical orientation and the root hair phenotype was
analyzed microscopically. A number of enl mutants were isolated and grown to the next generation. After confirmation of
the phenotypes in the enl/lrx1 double mutants, four lines (enl1,
enl2, enl5, enl7) displaying a range of different phenotypes
were characterized in more detail (Fig. 1).
Phenotypic analysis of the enl/lrx1 double mutants
The root hairs of enl1/lrx1 plants are shorter than wild
type, wavy, branch, and undergo random changes in the growth
direction. In addition, the root hair base is frequently larger
than in wild type. enl2/lrx1 plants develop root hairs that are
very short. Often, swollen bases develop and the hair proper is
almost absent. Similarly, root hairs of enl5/lrx1 plants are short
and frequently have enlarged root hair bases. enl7/lrx1 plants
develop a few root hairs that appear wild type like in length but
736
enl mutants enhance the lrx1 mutant phenotype
firming that the enl loci represent recessive loss-of-function
mutations.
Fig. 2 CAPS marker to distinguish the LRX1 from the lrx1-1s allele.
A fragment encompassing the 6-bp deletion in the lrx1-1s allele was
amplified by PCR and digested with the restriction enzyme BsiEI. The
wild-type LRX1/LRX1 genotype (left lane) gives two, the lrx1-1s/lrx11s (middle lane) three and a heterozygous plant four bands (right lane).
often branch at the enlarged bases, whereas many root hairs
develop into short stumps. In most cases, these stumps have an
enlarged root hair base.
As a first step in the genetic analysis, the enl lines were
backcrossed with the lrx1-1s mutant. The F1 seedlings of these
crosses all showed an lrx1 phenotype, indicating that the enl
mutations are recessive. The analysis of the F2 population
revealed a 1 : 3 segregation for enl/lrx1:lrx1 phenotypes (confirmed by a χ2 test, 5% error probability; data not shown) con-
enl phenotypes in the single mutant background
In a next step, we wanted to analyze whether the enl
mutant phenotypes are dependent on the mutant lrx1/lrx1
locus. An enl locus that would strongly enhance the lrx1 phenotype in an enl/lrx1 double mutant but would not cause an
obvious phenotype as an enl single mutant would indicate synergistic interaction between the two loci and thus suggest a
function in an LRX1-related process. To answer this question,
the enl lines (enl/enl; lrx1-1s/lrx1-1s) were crossed with wildtype Columbia (LRX1/LRX1). The F2 progeny segregated for
wild type, lrx1, enl/lrx1 and an additional fourth phenotype.
This latter phenotype was clearly distinguishable from the enl/
lrx1 double mutant for enl2, enl5, and enl7, but quite similar in
the case of enl1. Plants displaying the additional fourth phenotype were selected as they were assumed to be enl/enl single
mutants. These lines were tested for the lrx1-1s mutation by a
cleaved amplified polymorphism (CAPS) marker. A PCR fragment encompassing the 6-bp deletion in lrx1-1s was produced
and digested with BsiEI (Fig. 2). While wild-type LRX1/LRX1
plants give two bands, the lrx1-1s mutation introduces an additional BsiEI site, resulting in three bands. Correspondingly, a
heterozygous line gives rise to all four bands. A large number
of selected putative enl/LRX1 lines were analyzed with this
CAPS marker and only lines with a wild-type LRX1/LRX1
background were propagated to the next generation. A
Fig. 3 Phenotypes of the enl single mutants. Mutant enl seedlings in the wild-type LRX1/LRX1 background were grown for 4 d. Only the root
tissue is shown. (A) Overview on the root hair phenotypes. (B) Enlargement of the root hairs. Col, wild-type Columbia. (A) Bar = 500 µm; (B)
Bar = 250 µm.
enl mutants enhance the lrx1 mutant phenotype
737
Table 1 Root hair length and width of the enl single mutants
Genotype
Wild-type Columbia
enl1
enl2
enl5
enl7
a
Root hair length (µm ± SD)
Root hair width (µm ± SD)
530±100
n.d. a
110± 30
180± 40
540±110
16±1.3
29±6.1
26±2.7
16±2.6
29±3.6
n.d. due to the wavy shape of the root hairs in enl1, their length was not determined.
Table 2 Root hair length of the different actin2 mutants
Genotype
Wild-type Columbia
der1-1
der1-2
der1-3
enl2
Root hair length of root (µm ± SD)
Root hair length of collet (µm ± SD)
596±103
193± 36
64± 9
56± 15
137± 30
605±116
293± 72
88± 44
70± 11
249± 62
homozygous LRX1/LRX1 line could be identified for all lines
except enl5. For enl5, only one heterozygous line was found,
which was then propagated to the next generation. There, an
enl5 line with the wild-type LRX1/LRX1 genotype was identified.
The root hair phenotype of the enl lines (enl/enl; LRX1/
LRX1) was then analyzed in detail (Fig. 3, Table 1). Similar to
enl1/lrx1, the enl1 single mutant forms root hairs that branch
from the enlarged base or the hair proper and seem to randomly change growth direction. enl2 develops root hairs that
are very short and often have a radially enlarged root hair
proper. This enlargement, however, is not confined to the root
hair base, as observed in the enl2/lrx1 double mutant, but
extends further to the tip (see also Fig. 4C). The root hairs of
enl5 appear like the wild type except for their reduced length.
enl7 seedlings develop root hairs of wild-type length but with a
clearly increased diameter in the lower part of the root hair.
From this enlarged area, root hair branching, resulting in secondary root hairs protruding perpendicularly from the main
hair proper, was frequently observed. In summary, the enl single mutants all develop a mutant phenotype and, when combined with lrx1, the mutations display additive phenotypes.
Mapping of the enl mutations and allelism tests with known
mutants
Mapping populations were established for the enl mutants
(ecotype Columbia) by crossing them with Landsberg erecta.
F2 populations of these crosses were grown and 25 mutant F2
plants per line were selected for genetic mapping. enl1 was
mapped distal of ciw4 on chromosome 3 and enl2 was found to
be on the same chromosome, close to nga 162. enl5 is on the
top of chromosome 1, in the proximity of nga 63. This was
already indicated by the low frequency of recombination
between enl5 and lrx1, which is located on the BAC F21F1 on
chromosome 1. enl7 maps to the bottom of chromosome 5, distal of ciw10. From the map positions of enl1 and enl7, we conclude that they represent new genetic loci involved in root hair
development.
The map position of enl2 and its phenotype, which is similar to the previously identified actin2 mutant der1-1 (Ringli et
al. 2002), prompted us to cross these two mutants. The cross
revealed that enl2 is an additional actin2 allele. The short root
hair phenotype and the map position of enl5 indicated that enl5
might be allelic to axr1, an auxin-resistant mutant with pleiotropic effects on plant development (Lincoln et al. 1990). However, the similarity in the phenotypes of enl5 and axr1 is
restricted to root hairs. The two mutants were crossed with
each other, and the complementation test revealed that enl5 is
not allelic to axr1 and thus represents a new mutant locus.
The enl2 nonsense mutation represents a phenotypically weak
allele
To characterize the enl2 mutation in more detail, the
actin2 gene was isolated by PCR from the enl2/enl2 mutant
background and sequenced. A point mutation (C to T) was
found in the sequence of several independently obtained PCR
products which changes Glu356 to a stop-codon resulting in a
truncated ACTIN2 protein lacking 22 amino acids at the Cterminus (Fig. 4A). To compare the mutant phenotypes of enl2
and the three der1 alleles, seedlings of these lines were grown
in parallel. The root hair length and shape is most similar
between the weak der1-1 allele and enl2. In both lines, often a
large part of the root hair proper has an increased diameter
(Fig. 4B, C). der1-2 and der1-3 have clearly stronger phenotypes with only very short root hairs. The measurement of root
738
enl mutants enhance the lrx1 mutant phenotype
hair lengths of the root and the collet region of the different
lines confirmed this observation (Table 2). While root hairs of
der1-2 and der1-3 are very short, those of der1-1 and enl2 have
an intermediate length compared to wild type. The different
phenotypes of the der1 alleles, which are all miss-sense mutations (Fig. 4A), show that der1-1 is a hypomorph and encodes a
partially functional protein, while the ACTIN2 proteins
encoded by der1-2 and der1-3 might be entirely inactive or at
least strongly reduced in their activity. The similar phenotypes
of der1-1 and enl2 suggest that also enl2 is a hypomorph with
remaining ACTIN2 activity. To further investigate this point,
enl2 was crossed with the strong der1-2 allele and the phenotype of the F1 seedlings was analyzed. Root hairs of the root
(Fig. 4B) and the collet-region (Fig. 4D) of F1 plants are very
similar to those of parental enl2 plants while root hairs of
der1-2 plants are several times shorter. The enl2-like phenotype in the F1 plants reflects suppression of the der1-2 phenotype by enl2 and indicates that the truncated ACTIN2 of enl2 is
indeed partially active.
Discussion
An EMS mutagenesis screen based on the root hair mutant
lrx1 (Baumberger et al. 2001) was performed and lead to the
isolation of four enl mutants. The enl mutants are recessive and
three of them represent new loci involved in root hair development. They all show an independent phenotype, i.e. aberrant
root hair development in the wild-type LRX1/LRX1 background and the enl/lrx1 double mutants predominantly display
additive phenotypes. These characteristics indicate that the
processes affected by the enl mutations function independently
of LRX1.
Different processes are disturbed in the enl mutants. enl1
seedlings develop short, wavy root hairs that frequently and
randomly change growth direction. This indicates a strongly
aberrant tip-growth process. Wavy root hairs were observed
after disturbing the Ca2+ gradient (Bibikova et al. 1997) or
treatment with microtubule-interfering drugs (Bibikova et al.
1998). However, in these cases, the general growth direction is
largely maintained, which is clearly not the case in enl1. In
Fig. 4 The enl2 line is mutated in the actin2 gene. The actin2 gene of
enl2 was amplified by PCR and sequenced. (A) The point mutations of
enl2 and of the previously identified actin2 alleles der1-1, der1-2, and
der1-3 (Ringli et al. 2002) are indicated in the model of the actin protein structure. The affected amino acid residue is given followed by the
new amino acid (der1 alleles) or the stop codon (enl2). TMR (tetramethylrhodamine-5-maleimide) shown in the right lower part was used to
facilitate crystallization (Otterbein et al. 2001). Comparison by light
microscopy (B and D) and DICM (C) of wild-type, enl2, the der1 alleles, and der1-2 × enl2 F1 seedlings reveal an intermediate phenotype of
enl2 and the F1 seedlings, comparable to der1-1. Root hairs of the root
(B and C) and the collet region (D) are shown. The intermediate phenotype of the F1 seedlings suggests a partially functional ACTIN2 in
enl2 plants. Col, wild-type Columbia. (B) Bar = 500 µm; (C) Bar =
50 µm; (D) Bar = 250 µm.
enl mutants enhance the lrx1 mutant phenotype
contrast, plants transformed with a constitutively active ROP2
(RHO-related GTPase) construct, or mutants in the rhd3 locus,
which encodes a GTP-binding protein, develop root hairs similar to the enl1 phenotype (Wang et al. 1997, Jones et al. 2002).
Thus, the similarity of these latter phenotypes with enl1 suggests that ENL1 might rather function in a GTP-mediated
process. The enl1 mutant fits in the same group as cen1, cen2,
and cen3, which have short root hairs combined with additional deformations (Parker et al. 2000). The actin2 mutant
enl2 is similar to previously isolated actin2 mutants (Ringli et
al. 2002, Gilliland et al. 2002). It is affected in tip growth but in
addition develops enlarged root hair bases, which fits well with
the proposed function of the actin cytoskeleton during root hair
development. As the actin cytoskeleton is involved in the vesicle transport important for tip growth (Cai et al. 1997, Staiger
2000), interference with this structure by a mutation in an actin
protein can be expected to result in uncontrolled trafficking and
deposition of vesicles during cell expansion, causing the
observed phenotype. Different experiments showed that
ACTIN2 is not the only functional ACTIN protein in root hairs.
Expression studies have shown that ACTIN8 is also expressed
in root hairs and that act2/act7 double mutants develop a severe
root hair phenotype, which is stronger than any act2 single
mutant phenotype, indicating a function of ACTIN2 and
ACTIN7 in the same process (An et al. 1996, Gilliland et al.
2002). Also, the F-actin structure of act2 knock-out mutants is
not significantly different from wild type (Gilliland et al. 2002,
Nishimura et al. 2003), confirming that in the absence of
ACTIN2 other ACTIN proteins maintain the actin cytoskeleton.
A useful tool for the analysis of redundant genes is the isolation
of dominant mutations. The recently isolated dominant mutant
act2-2D shows severely aberrant root hairs, a general deficiency in plant development, and, on the cytological level, short
F-actin bundles (Nishimura et al. 2003). This is well in line
with the other data that propose a function of ACTIN2 in the
formation of actin filaments in root hair development. This
study shows that ACTIN2 is also expressed and important in
other tissues where no mutant phenotype can be observed in
the recessive loss-of-function act2 mutants and demonstrates
the strength of dominant mutations in the elucidation of gene
function. enl5 root hairs appear normal beside the strongly
reduced length of the hair proper, indicating that the mutation
is different from enl1 or enl2. Based on its phenotype, enl5 is
similar to bst1 (Parker et al. 2000) and might thus be distorted
by the same mechanism, which possibly affects rate and/or
duration of root hair elongation. In contrast to enl1, enl2, and
enl5, the mutant enl7 seems not generally affected in tip
growth. The length of enl7 root hairs is comparable to wild
type and the mutant phenotype is restricted to the lower root
hair proper that has an increased diameter. In addition, secondary root hairs form as branches from the primary root hairs.
This phenotype can be explained by a defective transition
phase between bulge formation and tip growth. The tip-growth
machinery in enl7 is presumably not well focused on a
739
restricted area on the bulge. This leads to an increased root hair
diameter and frequently to the installation of two tip-growth
machineries, resulting in the formation of branches. Subsequently, the tip-growth process is focused and the main hair
proper gains the correct diameter and length and thus a wildtype appearance, while the secondary root hair remains short.
In the lrx1/lrx1 mutant background, the enl mutants show
additive phenotypes. enl1/lrx1 plants develop wavy root hairs
with dramatic changes in growth direction and frequently
enlarged root hair bases. This suggests that the random growth
process displayed in enl1 is independent of LRX1. The enl2/
lrx1 and enl5/lrx1 double mutants support the conclusion made
of the respective single mutant phenotypes. enl2/lrx1 plants
mostly develop spherical structures at the root hair base with
barely detectable hairs, whereas enl5/lrx1 plants show a phenotype similar to lrx1 combined with the short root hairs characteristic for enl5. enl7/lrx1 plants form many short stumps, an
effect that can be attributed to the lrx1 mutation. In addition,
the root hair bases are enlarged although not to the same extent
as in enl2/lrx1 plants and branched root hairs develop.
The point mutation in enl2 introduces a stop codon at
Glu356 and thus truncates the ACTIN2 protein at the Cterminus by 22 amino acids. It appears that the C-terminus is
not absolutely vital for ACTIN2 function as the enl2 line seems
to encode a partially active ACTIN2. Both microscopic analysis and length measurement of the root hairs show that the enl2
phenotype is similar to der1-1 but less pronounced than the
previously isolated der1-2 and der1-3 mutants (Ringli et al.
2002). F1 seedlings of the cross enl2 × der1-2 have a phenotype that is closer to enl2 than der1-2, indicating a partial suppression of the der1-2 phenotype. Several amino acids within
the domain missing in enl2 are well conserved among the
Arabidopsis ACTIN family and thus evolutionary stable
(McDowell et al. 1996). This points towards a function in the
dynamics of actin cytoskeleton formation or one of the many
processes that require an actin cytoskeleton (Staiger 2000).
Indeed, among the amino acids missing are those involved in
profilin binding (Schutt et al. 1993) and actin–actin interaction
during actin filament formation (Holmes et al. 1990). Profilin
binds actin monomers and is important for the dynamics of
actin sequestration and polymerization (Staiger 2000). Interfering with this process is likely to influence actin filament formation. It can be assumed that the binding capacity of the
truncated actin within the actin filament or with profilin is
affected, leading to the observed phenotype.
In conclusion, we have characterized four recessive enl
mutations that are affected in root hair development and show
additive effects in combination with the lrx1 mutation. Three of
the four enl mutants represent new genetic loci involved in this
process. To functionally characterize root hair development, it
is necessary to saturate the process with mutations. The isolation of new root hair mutants reveals that we are still far from
saturation and additional efforts are required to achieve this
goal. Interestingly, molecular and phenotypic comparison of
740
enl mutants enhance the lrx1 mutant phenotype
several actin2-alleles indicates that the deletion of 22 amino
acid residues at the C-terminus is not deleterious for ACTIN2
function despite previous findings that this domain is involved
in protein–protein interactions. A more detailed characterization of the enl2 line should reveal the function in which this
mutant is affected and thus provide insight into the process the
C-terminus of ACTIN proteins is involved in.
Materials and Methods
Plant material and growth conditions
Seeds of the Arabidopsis line (ecotype Columbia) containing the
stable lrx1-1s allele described in the Results section was EMSmutagenized and grown to maturity in soil under greenhouse conditions. Seeds were surface sterilized for 10 min with 1% Na-hypochlorite, 0.03% (v/v) Triton X-100, washed three times with sterile water
and stratified for 3 d at 4°C. Subsequently, they were grown on halfstrength MS-medium, 2% sucrose, 0.6% Phytagel (Sigma, Buchs,
Switzerland) in Petri plates in a vertical orientation for 4 d at 24°C
under continuous light for visual inspection of the root hair phenotype. The der1-1, der1-2, and der1-3 mutations are in the C24 background and were previously described (Ringli et al. 2002).
Microscopic analysis
For visual screening of the root hair phenotype, a Leica stereomicroscope MZ 125 (Leica, Glattbrugg, Switzerland) was used. Differential interference contrast microscopy (DICM) was performed on an
axioplan microscope (Zeiss, Jena, Germany). For measuring root hair
length and width, pictures from plant material were taken with the
DICM microscope, printed and then measured.
Molecular and genetic analysis
For mapping, seedlings of F2 populations of the crosses enl
(Columbia) × Landsberg erecta were grown as described and visually
selected. Twenty-five mutant F2 seedlings per population were selected
and genomic DNA was extracted according to Fulton et al. (1995). The
mapping was performed using standard CAPS and simple sequence
length polymorphism markers.
For sequencing of the actin2 gene of enl2, plant genomic DNA
was isolated according to Fulton et al. (1995). PCR was performed
using gene specific primer pairs. The PCR products were purified with
the GFX PCR purification kit (Amersham Biosciences, Otelfingen,
Switzerland) prior to sequencing.
Acknowledgments
We thank Joelle Sasse for technical assistance, Dr. Roberto
Dominguez for the permission to use the actin structure model, and Dr.
Beat Keller and Dr. Margaret Collinge for critical reading of the manuscript. This work was supported by the Swiss National Science Foundation (Grant Nr. 31–61419.00).
References
An, Y.Q., Huang, S.R., McDowell, J.M., McKinney, E.C. and Meagher, R.B.
(1996) Conserved expression of the Arabidopsis ACT1 and ACT3 actin subclass in organ primordia and mature pollen. Plant Cell 8: 15–30.
Baumberger, N., Doesseger, B., Guyot, R., Diet, A., Parsons, R.L., Clark, M.A.,
Simmons, M.P., Bedinger, P., Goff, S.A., Ringli, C. and Keller, B. (2003a)
Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis
and rice: a conserved family of cell wall proteins form a vegetative and a
reproductive clade. Plant Physiol. 131: 1313–1326.
Baumberger, N., Ringli, C. and Keller, B. (2001) The chimeric leucine-rich
repeat/extensin cell wall protein LRX1 is required for root hair morphogenesis in Arabidopsis thaliana. Genes Dev. 15: 1128–1139.
Baumberger, N., Steiner, M., Ryser, U., Keller, B. and Ringli, C. (2003b) Synergistic interaction of the two paralogous Arabidopsis genes LRX1 and LRX2 in
cell wall formation during root hair development. Plant J. 35: 71–81.
Bibikova, T.N., Jacob, T., Dahse, I. and Gilroy, S. (1998) Localized changes in
apoplastic and cytoplasmic pH are associated with root hair development in
Arabidopsis thaliana. Development 125: 2925–2934.
Bibikova, T.N., Zhigilei, A. and Gilroy, S. (1997) Root hair growth in Arabidopsis thaliana is directed by calcium and an endogenous polarity. Planta 203:
495–505.
Braun, M., Baluska, F., von Witsch, M. and Menzel, D. (1999) Redistribution of
actin, profilin and phosphatidylinositol-4,5-bisphosphate in growing and
maturing root hairs. Planta 209: 435–443.
Cai, G., Moscatelli, A. and Cresti, M. (1997) Cytoskeletal organization and pollen tube growth. Trends Plant Sci. 2: 86–91.
Dolan, L., Duckett, C.M., Grierson, C., Linstead, P., Schneider, K., Lawson, E.,
Dean, C., Poethig, S. and Roberts, K. (1994) Clonal relationships and cell
patterning in the root epidermis of Arabidopsis. Development 120: 2465–
2474.
Favery, B., Ryan, E., Foreman, J., Linstead, P., Boudonck, K., Steer, M., Shaw,
P. and Dolan, L. (2001) KOJAK encodes a cellulose synthase-like protein
required for root hair cell morphogenesis in Arabidopsis. Genes Dev. 15: 79–
89.
Foreman, J. and Dolan, L. (2001) Root hairs as a model system for studying
plant cell growth. Ann. Bot. 88: 1–7.
Foreman, J., Demidchik, V., Bothwell, J.H.F., Mylona, P., Miedema, H., Torres,
M.A., Linstead, P., Costa, S., Brownlee, C., Jones, J.D.G., Davies, J.M. and
Dolan, L. (2003) Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422: 442–446.
Fulton, T.M., Chunwongse, J. and Tanksley, S.D. (1995) Microprep protocol for
extraction of DNA from tomato and other herbaceous plants. Plant Mol. Biol.
Rep. 13: 207–209.
Gilliland, L.U., Kandasamy, M.K., Pawloski, L.C. and Meagher, R.B. (2002)
Both vegetative and reproductive actin isovariants complement the stunted
root hair phenotype of the Arabidopsis act2-1 mutation. Plant Physiol. 130:
2199–2209.
Grierson, C.S., Parker, J.S. and Kemp, A.C. (2001) Arabidopsis genes with roles
in root hair development. J. Plant Nutr. Soil Sci.-Z. Pflanzenernah. Bodenkd.
164: 131–140.
Holmes, K.C., Popp, D., Gebhard, W. and Kabsch, W. (1990) Atomic model of
the actin filament. Nature 347: 44–49.
Jones, M.A., Shen, J.J., Fu, Y., Li, H., Yang, Z.B. and Grierson, C.S. (2002) The
Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation
and tip growth. Plant Cell 14: 763–776.
Lincoln, C., Britton, J.H. and Estelle, M. (1990) Growth and development of the
axr1 mutants of Arabidopsis. Plant Cell 2: 1071–1080.
Masucci, J.D. and Schiefelbein, J.W. (1994) The rhd6 mutation of Arabidopsis
thaliana alters root hair initiation through an auxin-associated and ethyleneassociated process. Plant Physiol. 106: 1335–1346.
McDowell, J.M., Huang, S.R., McKinney, E.C., An, Y.Q. and Meagher, R.B.
(1996) Structure and evolution of the actin gene family in Arabidopsis thaliana. Genetics 142: 587–602.
Miller, D.D., de Ruijter, N.C.A., Bisseling, T. and Emons, A.M.C. (1999) The
role of actin in root hair morphogenesis: studies with lipochito-oligosaccharide as a growth stimulator and cytochalasin as an actin perturbing drug. Plant
J. 17: 141–154.
Nishimura, T., Yokota, E., Wada, T., Shimmen, T. and Okada, K. (2003) An
Arabidopsis ACT2 dominant-negative mutation, which disturbs F-actin
polymerization, reveals its distinctive function in root development. Plant
Cell Physiol. 44: 1131–1140.
Otterbein, L.R., Graceffa, P. and Dominguez, R. (2001) The crystal structure of
uncomplexed actin in the ADP state. Science 293: 708–711.
Oyama, T., Shimura, Y. and Okada, K. (2002) The IRE gene encodes a protein
kinase homologue and modulates root hair growth in Arabidopsis. Plant J.
30: 289–299.
enl mutants enhance the lrx1 mutant phenotype
Parker, J.S., Cavell, A.C., Dolan, L., Roberts, K. and Grierson, C.S. (2000)
Genetic interactions during root hair morphogenesis in Arabidopsis. Plant
Cell 12: 1961–1974.
Rigas, S., Debrosses, G., Haralampidis, K., Vicente-Agullo, F., Feldmann, K.A.,
Grabov, A., Dolan, L. and Hatzopoulos, P. (2001) TRH1 encodes a potassium
transporter required for tip growth in Arabidopsis root hairs. Plant Cell 13:
139–151.
Ringli, C., Baumberger, N., Diet, A., Frey, B. and Keller, B. (2002) ACTIN2 is
essential for bulge site selection and tip growth during root hair development
of Arabidopsis. Plant Physiol. 129: 1464–1472.
Schiefelbein, J.W. (2000) Constructing a plant cell. The genetic control of root
hair development. Plant Physiol. 124: 1525–1531.
Schutt, C.E., Myslik, J.C., Rozycki, M.D., Goonesekere, N.C.W. and Lindberg,
U. (1993) The structure of crystalline profilin beta-actin. Nature 365: 810–
816.
741
Seifert, G.J., Barber, C., Wells, B., Dolan, L. and Roberts, K. (2002) Galactose
biosynthesis in Arabidopsis: Genetic evidence for substrate channeling from
UDP-D-galactose into cell wall polymers. Curr. Biol. 12: 1840–1845.
Staiger, C.J. (2000) Signaling to the actin cytoskeleton in plants. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 51: 257–288.
Vandekerckhove, J.S., Kaiser, D.A. and Pollard, T.D. (1989) Acanthamoeba
actin and profilin can be crosslinked between glutamic acid 364 of actin and
lysine 115 of profilin. J. Cell Biol. 109: 619–626.
Wang, H.Y., Lockwood, S.K., Hoeltzel, M.F. and Schiefelbein, J.W. (1997) The
ROOT HAIR DEFECTIVE3 gene encodes an evolutionarily conserved protein with GTP-binding motifs and is required for regulated cell enlargement
in Arabidopsis. Genes Dev. 11: 799–811.
Webb, M., Jouannic, S., Foreman, J., Linstead, P. and Dolan, L. (2002) Cell
specification in the Arabidopsis root epidermis requires the activity of
ECTOPIC ROOT HAIR 3 – a katanin-p60 protein. Development 129: 123–
131.
(Received October 27, 2003; Accepted March 25, 2004)