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Plant Cell Physiol. 46(7): 1046–1053 (2005)
doi:10.1093/pcp/pci115, available online at www.pcp.oupjournals.org
JSPP © 2005
The Arabidopsis Root Hair Mutants der2–der9 are Affected at Different
Stages of Root Hair Development
Christoph Ringli * , Nicolas Baumberger 1 and Beat Keller
Institute of Plant Biology, University of Zürich, Zollikerstr. 107, 8008 Zurich, Switzerland
;
Root hairs are an excellent model system to study cell
developmental processes as they are easily accessible, single-celled, long tubular extensions of root epidermal cells.
In a genetic approach to identify loci important for root
hair development, we have isolated eight der (deformed root
hairs) mutants from an ethylmethanesulfonate (EMS)-mutagenized Arabidopsis population. The der lines represent five
new loci involved in root hair development and show a variety of abnormalities in root hair morphology, indicating
that different root hair developmental stages are affected. A
double mutant analysis with the short root hair actin2
mutant der1-2 confirmed that the der mutants are disturbed at different time points of root hair formation.
Auxin and ethylene are known to be important for trichoblast cell fate determination and root hair elongation. Here,
we show that they are able to suppress the phenotype of two
der mutants. As the auxin- and ethylene-responsive der
mutants are affected at different stages of root hair formation, our results demonstrate that the function of auxin and
ethylene is not limited to cell differentiation and root hair
elongation but that the two hormones are effective throughout the whole root hair developmental process.
Keywords: Auxin — der mutants — Ethylene — Root hairs.
Abbreviations: ACC, aminocyclopropane carboxylic acid; der,
deformed root hair; EMS, ethylmethanesulfonate.
Introduction
Root epidermal cells develop long thin extensions for
anchorage of the plant and to enlarge the root surface, thereby
increasing the efficiency of water and nutrient uptake from the
soil (Gilroy and Jones 2000). These root hairs are long tubular
structures formed by a polarized outgrowth at the apical end of
epidermal cells. The root hair structure is easily accessible and
can be microscopically analyzed without disturbing the surrounding tissue, which makes it an excellent model system to
study cell growth and development (Foreman and Dolan 2001).
In Arabidopsis, epidermal cells differentiate into root hairforming cells (trichblasts) or non-root hair-forming cells
(atrichblasts) depending on positional cues relative to the
underlying cortex cells. Trichoblasts form when the epidermal
1
*
cell is in contact with the anticlinal wall of two adjacent cortical cells, whereas atrichoblast cells are located over the perclinal wall of only one cortex cell (Dolan et al. 1994). The
developmental divergence of the two types of root epidermal
cells can be observed long before the outgrowth of the hair
proper by a denser cytoplasm and a higher vacuolation rate in
trichoblast cells and a generally longer cell shape of the
atrichoblasts (Dolan et al. 1994, Galway et al. 1994). In
Arabidopsis, several genes such as WER, TTG, GL2, CPC,
RHL1-3 and ERH3 have been implicated in root epidermal cell
patterning as mutations at these loci lead to either ectopic root
hair formation or a strongly reduced number of root hairs
(Rerie et al. 1994, Wada et al. 1997, Schneider et al. 1997, Lee
and Schiefelbein 1999, Walker et al. 1999, Webb et al. 2002).
Once trichoblasts are set to form a root hair, this developmental process can be divided into three stages. After the formation of a bulge at the apical end of the trichoblast, the root hair
proper starts to elongate by slow tip growth. In a last step, the
transition to an accelerated tip growth leads to the formation of
the fully extended root hair (Dolan et al. 1994).
The phytohormones auxin and ethylene have a strong
effect on epidermal cell differentiation. In sufficient concentration, the ethylene precursor aminocyclopropane carboxylic acid
(ACC) induces the formation of root hairs at ectopic positions,
while application of the ethylene biosynthesis inhibitor AVG
[L-α-(2-amino-ethoxyvinyl)-glycine] prevents development of
root hairs (Tanimoto et al. 1995). In addition, ACC induces
root hair formation in the root hair-less mutant rhd6 (Masucci
and Schiefelbein 1994). The same study showed that the auxin
IAA has an identical effect on rhd6. Auxin and ethylene are
important not only for cell patterning, but also for root hair formation as the auxin response mutants aux1 and axr1 and the
ethylene mutants ein2 and etr1 all show reduced root hair
length (Pitts et al. 1998). On the cellular level, ethylene participates in the flux control of nucleotide sugars, modifies the
composition of cell wall polymers, and thus directly affects the
structure of the extracellular matrix (Seifert et al. 2004).
Several morphological root hair mutants have been
described that show aberrant root hair formation (for a review,
see Grierson et al. 2001). Common features of these mutants
are root hairs that are short, wavy, branched, increased in diameter, or form bulbous structures at the base of the root hair. The
root hair bases of the mutants rhd1, lrx1 and kjk are enlarged
Present address: Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Corresponding author: E-mail, [email protected]; Fax, +41-44-634-82-04.
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Arabidopsis der (deformed root hair) mutants
1047
Fig. 1 Root hair phenotype of der mutants.
Arabidopsis seedlings were grown in a vertical
orientation for 5 d on half-strength MS medium
and observed under a stereomicroscope (A).
Arrowheads indicate examples of spherical structures at the root hair base. (B) Scanning electron
microscopic pictures of root hairs of wild-type,
der3, der7 and der8 lines to show root hair
branching (der3), the act2-like phenotype of der7
and the swollen and burst root hairs of der8
plants. Arrows indicate branched root hairs in
der3 and burst root hairs in der8. (A) Scale bar =
300 µm. (B) Scale bar = 100 µm.
and often collapse. This is probably caused by a weakened cell
wall as these genes encode proteins presumably involved in
cell wall biosynthesis or control of cell wall architecture
(Baumberger et al. 2001, Favery et al. 2001, Seifert et al. 2002,
Baumberger et al. 2003, Ringli 2005). Interestingly, the rhd1
mutant phenotype is suppressed by ethylene, probably due to
its effect on the nucleotide sugar flux and, as a consequence, on
the cell wall structure (Seifert et al. 2004). Defects in the tip
growth important for root hair elongation (Gilroy and Jones
2000) result in short root hairs. The Ca2+ gradient required for
this process (Bibikova et al. 1997) is absent in root hairs of
rhd2 plants which is due to a mutation in an NADPH-oxidase
involved in the regulation of Ca2+ channel activity (Foreman et
al. 2003). Mutations in the K+ transporter TRH1 and a protein
kinase also lead to short root hairs (Rigas et al. 2001, Oyama et
al. 2002). The actin cytoskeleton is crucial for root hair growth,
as shown by studies with actin-interfering chemicals as well as
the characterization of the ACTIN2 mutant der1 (Miller et al.
1999, Ringli et al. 2002). tip1, cow1, shv1, shv2 and shv3 are
involved in the early stages of tip growth, while bst1, cen1,
cen2, cen3 and scn1 seem to act later in the same process
(Parker et al. 2000). Wavy root hairs are caused by mutations in
the rhd3 locus coding for a GTP-binding protein (Wang et al.
1997) or by interfering with microtubule formation by cytochemical compounds (Bibikova et al. 1999).
Here, we describe deformed root hairs 2–9 (der2-9) a
series of Arabidopsis root hair mutants of which five represent
new loci involved in root hair development. The mutant lines
were isolated in a screen of ethylmethanesulfonate (EMS)mutagenized plants for aberrant root hair development. Microscopic analysis of der single and double mutants indicates that
the der mutants are affected at different stages of root hair
development, leading to a variety of mutant phenotypes.
Recently, Seifert et al. (2004) have shown that ethylene suppresses the morphological root hair mutant rhd1 by altering the
polysaccharide composition of the cell wall. We demonstrate
that ethylene and auxin suppress two der mutants that are
affected at different stages of development. This indicates that
auxin and ethylene participate in root hair formation throughout the whole developmental process.
Results
Isolation of der mutants
EMS mutagenesis was performed on Arabidopsis seeds of
the ecotype C24 and mutagenized plants were grown to the M2
generation in 12 pools of 1,000 plants each. M2 seeds were
grown on MS plates, and microscopic analysis of the root hairs
was performed to select mutant lines. The examination of
70,000 M2 seedlings led to the isolation of >50 mutant lines
that displayed aberrant root hair morphology. Nine of these
lines were subsequently characterized in more detail and
named deformed root hairs 1–9 (der1-9; Ringli et al. 2002 and
this work). Backcrossing of the mutants with wild-type C24
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Arabidopsis der (deformed root hair) mutants
Table 1 Segregation analysis of the der mutant phenotypes in
an F2 population
Segregating No. of seeds % of mutant χ2
F2der×C24 analyzed
value a Segregation
seedlings
der2
der3
der4
der5
der6
der7
der8
der9
a
603
473
285
462
380
377
361
319
19
25
26
22
18
28
20
23
11.3
0.04
0.42
2.10
9.49
1.63
4.39
0.55
Distorted
Normal
Normal
Normal
Distorted
Normal
Distorted
Normal
The χ2 value is calculated based on a 3 : 1 ratio of wild-type to mutant
plants. The critical value is 3.84 (P = 0.05).
resulted in F1 seedlings displaying a wild-type phenotype. The
frequencies of der mutant phenotypes in the segregating F2
populations indicated that all der mutants are recessive and
thus loss-of-function mutations. Complementation tests among
the isolated mutants did not lead to the isolation of independent alleles of the same mutation.
Morphological analysis of der mutants
The der mutants display an array of different phenotypes.
Mutants of one group (der2, der5, der6, der8 and der9) often
display very short stumps or collapsed root hairs (Fig. 1a).
Among them, der2 and der9 have mostly very short root hairs,
indicating a defect in the transition to rapid tip growth, which is
required to fully develop the hair structure. Root hairs of der5,
der6 and der8 are often wavy, have an irregular hair diameter
or are collapsed. In addition, der5 and der8 sometimes develop
bulbous structures at the root hair base reminiscent of the lrx1
mutant phenotype (Fig. 1b; Baumberger et al. 2001). In contrast, seedlings of the second group of der mutants (der3, der4
and der7) form root hairs in a regular pattern (Fig. 1a). In der3,
the length of the hair proper is not affected, but side branches
develop from the enlarged bases (Fig. 1b). Root hairs of der4
and der7 are very regular but significantly shorter than in the
wild type. While der4 appears normal beside the reduced
length, der7 develops two different types of root hair structures: relatively short stumps with a clearly wider diameter
than wild-type hairs and longer hair structures which are wider
in the lower half of the root hair proper while the upper half is
wild type like (Fig. 1b). The der7 phenotype is thus highly similar to the weak actin2 allele der1-1 (Ringli et al. 2002), which
suggests a possibly similar function for the two loci.
Distorted segregation due to der mutations
Root hairs and pollen tubes develop by tip growth, and
factors involved in this process potentially function in both cell
types. Thus, genetic loci involved in root hair formation at the
same time can have a deleterious effect on pollen development
Fig. 2 Root hair phenotypes of double mutants. rhd1, rhd2 and the
der mutants were crossed with der1-2, a strong actin2 allele, and double mutants were analyzed in the F2 population. rhd1 and rhd2 were
used as controls as they are known to be affected very early during
bulge formation and later in the tip growth process, respectively, and
thus cause very different root hair phenotypes in combination with
der1-2. der3, der5 and der8 cause bulbous structures in the double
mutants whereas all other double mutants display the der1-2 phenotype (not shown). Scale bar = 300 µm.
(Schiefelbein et al. 1993). For this reason, pollen fitness was
measured by analyzing the inheritance of the der mutations. To
this end, the frequencies of the der mutant phenotypes in segregating F2 populations of the third backcross into wild-type C24
were investigated (Table 1). The statistical analysis by the χ2
test revealed that der2, der6 and der8 show a frequency of the
wild-type vs mutant phenotype that is significantly different
from the 3 : 1 ratio expected for a recessive mutation. One possible explanation for the reduced transmission of the mutations
to the next generation is an effect on pollen viability and thus a
general role for a subset of the der loci in tip growth-related
processes. The other der mutants did not show a significant
deviation from the 3 : 1 segregation, indicating that the effect
of these mutations is root hair specific.
The der mutations act at different developmental stages during
root hair development
Double mutants of the der mutants with der1-2, a strong
actin2 allele that shows very short root hairs (Ringli et al.
2002), were generated to position the function of the der loci
during root hair development (Fig. 2). der1-2 is affected after
root hair initiation, mainly in the tip growth process. Thus,
der1-2 can be expected to be epistatic to mutations affected in a
Arabidopsis der (deformed root hair) mutants
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Table 2 Length of fully grown root hairs on control or hormone-containing growth medium
Line
Control
(mm ± SD)
ACC
(mm ± SD)
IAA
(mm ± SD)
C24
der 8
der 9
0.77 ± 0.21
0.35 ± 0.11
0.30 ± 0.08
1.11 ± 0.16
0.57 ± 0.15
0.55 ± 0.10
0.90 ± 0.13
0.61 ± 0.10
0.52 ± 0.11
For the analysis, five seedlings were used and 20 root hairs of each
were measured. In der8 and der9, collapsed root hairs and short stumps
(absence of tip growth) were not included in the measurement.
Double mutants with der2, der4, der6, der7 and der9 were
indistinguishable from the der1-2 single mutant (data not
shown). Hence, the der genes affected in the latter mutants
appear to act later in root hair development, during tip growthrelated processes.
Fig. 3 Hormone treatment of der mutants. der seedlings were grown
for 4 d on MS medium, transferred onto non-supplemented control MS
medium (left column), MS medium containing 5×10–7 ACC (right column) and 5×10–8 IAA (middle column), respectively, and grown for an
additional 4 d. As a positive control for the experimental set-up, root
hair induction by ACC and IAA in the root hair-less mutant rhd6 was
successfully tested. der mutants with similar deficiencies in root hair
development (der2, der5, der6, der8 and der9) show different
responses to the phytohormones. Scale bar = 300 µm.
late stage of root hair development, i.e. corresponding double
mutants are likely to display a der1-2 phenotype. In contrast,
mutations affected early in root hair development should either
cause an additive phenotype in the double mutant or be epistatic to der1-2. In a test experiment, double mutants were
established of der1-2 with rhd1, a mutant affected in cell wall
extension early during root hair development, and rhd2,
involved in tip growth and thus later in the developmental
process (Andème-Onzighi et al. 2002; Foreman et al. 2003). As
predicted, root hairs of rhd1 der1-2 double mutants developed
an additive phenotype with large bulges and absent root hairs,
whereas, in rhd2 der1-2 plants, the der1-2 phenotype was epistatic to rhd2. This supports our assumption that der1-2 is an
excellent tool to assign the effect of a mutation to a phase in
root hair development.
The mutants der3, der5 and der8, in combination with
der1-2, caused the formation of bulbous structures (Fig. 2),
revealing that these mutants are affected early during root hair
development. Branching of root hairs observed in der3 seedlings was not detected in the double mutant, indicating that initiation of tip growth is required for branching to take place.
The der mutants show distinct responses to auxin and ethylene
The application of exogenous auxin and ethylene has been
shown to suppress aberrant root hair development of several
auxin and ethylene mutants (Masucci and Schiefelbein 1994,
Pitts et al. 1998). Ethylene also changes the nucleotide sugar
flux in the cell which influences the composition of the extracellular matrix and suppresses rhd1, a root hair mutant affected
in the composition of the cell wall (Seifert et al. 2004). To
investigate whether the morphological root hair mutants
described here are rescued by the application of exogenous ethylene and/or auxin, mutant seedlings were germinated and
grown for 4 d on MS medium, then transferred to unsupplemented control or hormone-containing medium, respectively,
and grown for an additional 4 d. As a positive control for the
activity of the supplied hormones, induction of root hair development in the normally root hair-less mutant rhd6 was tested
which showed a strong response to both hormones (data not
shown). Wild-type root hairs showed a slight increase in length
upon hormone treatment, confirming that ACC and IAA
enhance tip growth (Fig. 3 and Table 2). The hormone treatments resulted in little or no effect on most der mutants. In
contrast, der8 and der9 showed a suppressed phenotype. In the
control experiment, both mutants developed few and often distorted root hairs. In the presence of auxin or ethylene, the seedlings displayed regular root hairs that were rarely misshapen
(Fig. 3). The bulges often found at the root hair bases in der8
control seedlings were not observed in the presence of the hormones. The length of the root hairs was also increased compared with the der8 and der9 mutants on control medium, even
though they did not reach wild-type length (Table 2). Hence,
two der mutants that are affected in different phases of root
hair development are responsive to auxin and ethylene treatment. Together with the finding that the effect of the rhd1
mutation, which is important for early stages of root hair formation, is suppressed by exogenously applied ethylene (Seifert
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Arabidopsis der (deformed root hair) mutants
Fig. 4 Map positions of the different der mutants. The der mutants
were mapped using 50 mutant plants of a segregating F2 population.
The loci are indicated on the five Arabidopsis chromosomes. Where
allelism to previously identified mutants was found, the name of the
allelic mutation is also given.
et al. 2004), this suggests that ethylene and auxin influence root
hair formation throughout the whole developmental process.
Mapping and allelism tests
The different der mutations (ecotype C24) were mapped
using segregating F2 populations of crosses with Columbia and
Landsberg erecta (Ler). Fifty mutant F2 plants were used for
mapping of each der locus. Fig. 4 shows the five chromosomes
of Arabidopsis with the genetic loci indicated. der2 and der9
showed no recombination with a newly established marker on
the TAC K21H1 on chromosome 5, between the markers ciw9
and ciw10. Their genetic proximity required an allelism test,
which revealed that the two mutations are not allelic. der4
showed one recombinant chromosome with ciw10, while der3
is further distal of ciw10 (seven recombinations in 50 plants).
der5 maps on chromosome 4, 10 cM proximal of nga1107.
der6 is located on chromosome 3, close to nga172. der7 maps
1 cM distal of nga1126 on chromosome 2 and der8 1 cM distal
of ciw4 on chromosome 3.
The proximity of der2, der3, der4, der5, der6 and der9 to
regions of the genome also identified for other root hair
mutants made corresponding allelism tests necessary. der2,
der4 and der9 were crossed with bst1 and shv2 (Parker et al.
2000), der3 with enl7 (Diet et al. 2004), der5 with cow1
(Grierson et al. 1997) and der6 with cen3, shv1 and scn1
(Parker et al. 2000). The complementation tests revealed that
der3/enl7, der4/bst1 and der9/shv2 are allelic while all other
der mutants constitute new genetic loci involved in root hair
development.
Discussion
Root hairs are an excellent system to study growth and
development of a single cell. To identify factors that are impor-
tant for root hair growth, an EMS-mutagenized Arabidopsis
population was screened for plants that display aberrant root
hair development. Eight der mutants were identified, of which
five represent new loci while three were found to be allelic to
the previously identified loci bst1, shv2 and enl7 (Parker et al.
2000, Diet et al. 2004). Despite the fact that a considerable
number of root hair mutants have been isolated (for review, see
Grierson et al. 2001), the collection of mutants is obviously far
from saturated as alleles of only a few previously identified
mutants were isolated. This is possibly due to the fact that root
hairs are isolated cells where even slightly aberrant development is easily detectable, enabling the isolation of a large
number of mutations causing detectable effects. Alternatively,
functional redundancy among genes expressed in root hairs
might be less common as they are not absolutely essential for
plant survival and therefore are under less stringent selection.
Pollen tubes represent a second cell type that develops by tip
growth (Cai et al. 1997). However, in contrast to root hairs,
pollen tubes are essential for plants as they are required for
reproduction. It has been revealed in the past that the growth
processes of the two cell types share many components, among
them a Ca2+ gradient, phosphatidylinositol-4,5-bisphosphate
and Rop GTPases (Pierson et al. 1996, Bibikova et al. 1997,
Braun et al. 1999, Kost et al. 1999, Jones et al. 2002). Hence, it
can be expected that at least a subset of the genes important for
root hair development have the same function in pollen. To
investigate the viability of pollen, measuring the frequency of
the mutant phenotype in a segregating F2 population is common practice. Distortion of the segregation ratio indicates
impaired transmission of the mutation through pollen. A second possible explanation for a distorted segregation would be
an impaired female gametophytic development. Considering
the similarities between root hair and pollen tube growth, the
latter possibility is certainly less likely. Among the 20 root hair
mutants for which transmission of the mutation was investigated (Schiefelbein et al. 1993, Grierson et al. 1997, Parker et
al. 2000, Rigas et al. 2001, Ringli et al. 2002, this study), a
clear effect was observed in tip1 (Schiefelbein et al. 1993),
der2, der6 and der8 (this study), i.e. 20% of the cases. This
supports the assumption that the tip growth machinery of root
hairs and pollen tubes indeed share some components.
der mutants are affected throughout root hair development
The der mutants are affected at different stages of root
hair development (Fig. 5). All of them are able to develop at
least to the bulge stage, the first step in the formation of the
hair proper (Dolan et al. 1994). Based on the data of the double mutant analysis with der1-2, a strong actin2 mutant that is
mainly deficient in tip growth (Ringli et al. 2002), der3/enl7,
der5 and der8 are affected in bulge formation. These double
mutants develop spherical structures at the root hair bases. This
additive phenotype, caused by the combination of an enlarged
initial bulge and a deficient tip growth, confirms that the der3/
enl7, der5 and der8 loci are involved in an early process that
Arabidopsis der (deformed root hair) mutants
Fig. 5 Schematic representation of der functions during root hair formation. The drawing indicates the different stages of root hair development, including cell type differentiation and root hair formation.
Below, vertical lines indicate the phases in which the different der loci
are involved in this process, based on their phenotype and the double
mutant analysis with the actin2 mutant der1-2. der1-2, rhd1, rhd2 and
rhd6, the previously described mutants used in this study (Ringli et al.
2002, Foreman et al. 2003, Seifert et al. 2004), are also listed (gray
boxes). Asterisks indicate suppression of the mutant phenotypes by
ethylene and auxin.
acts before tip growth initiates and are important to limit the
initial bulge formed on the epidermis. Bulbous structures can
result from a weak extracellular matrix causing extensive wall
enlargement, as has been shown for rhd1 and lrx1 that are
involved in the biosynthesis of cell wall components and the
establishment of its architecture, respectively (Seifert et al.
2002, Baumberger et al. 2003). Hence, based on their phenotypes, der3/enl7, der5 and der8 might be involved in cell wall
development. Alternatively, these loci might be important to
focus the tip growth machinery on the initial bulge, and their
absence might induce an uncontrolled expansion of the bulge.
At least in der3, this deficiency would be temporary as, after
the development of an enlarged root hair base, a root hair of
normal length develops. The next step of root hair formation is
the initiation of the tip growth machinery at the defined point
on the bulge (Dolan et al. 1994). This step is affected in der2,
der5, der6, der7, der8 and der9/shv2, which is observable by
short root hair initials that do not elongate, or, for der7, by
larger root hair propers. In the latter mutant, the tip might be
limited to a somewhat larger area on the initial bulge, leading
to cell growth in a wider region of the tip. In the double
mutants with der1-2, der2, der6, der7 and der9/shv2 do not
cause a swelling of the initial bulge, supporting the assumption
that they are affected after der1-2, i.e. in a later phase of root
1051
hair development. Eventually, the root hair enters the final
phase when rapid elongation by tip growth takes place (Dolan
et al. 1994). der4/bst1 and der7 are affected in this phase of
development as their root hairs are shorter than in wild type. As
root hairs of der7 are also enlarged in diameter, an uncontrolled trafficking and deposition of cell wall material-containing vesicles during tip growth (Cai et al. 1997) might be the
cause of the aberrant development. This phenotype is reminiscent of der1-1, a weak actin2 mutant (Ringli et al. 2002) and
can also be observed after treatment of root hairs with actin
cytoskeleton-interfering drugs (Miller et al. 1999). Thus, the
DER7 locus might function in a process related to the actin
cytoskeleton. From the map position of der7, a mutation in a
second actin gene can be excluded (McKinney and Meagher
1998). Hence, map-based cloning of der7 will be necessary to
verify this hypothesis.
The branched root hair phenotype of der3/enl7 indicates
that beside bulge formation, der3/enl7 has a second effect in a
later phase of root hair development. Whether the two distinct
phenotypes are both a direct cause of the der3/enl7 mutation or
whether the branching of root hairs is a consequence of the
enlarged hair proper remains to be determined. The latter case
would suggest that there are different types of ‘enlarged bases’
as not all mutants with this phenotype also develop branched
root hairs. Additional evidence for different ways for a trichoblast to develop enlarged bases arises from the analysis of the
der double mutants with der1-2. The enlarged bases found in
the mutant lines der3/enl7, der5, and der8 are also present in
the double mutant background with der1-2. In contrast, der1-2
is clearly epistatic to der7, which by itself also develops root
hair bases with an increased diameter. Thus, a different functional principle leading to an enlarged hair base is affected in
der7 plants.
Suppression of a subset of der mutations through auxin and ethylene
The effect of ACC and IAA on root hair development was
investigated by growing the der mutants on hormone-supplemented media. der8 and der9/shv2 show a strong response to
both ACC and IAA, whereas the phenotypes of the other der
mutants were not suppressed. Both the shape and the length of
the root hairs in der8 and der9/shv2 were strongly modified by
ACC and IAA. Thus, auxin and ethylene not only suppress root
hair initiation and elongation mutants such as rhd6, ein2, etr1
and aux1 (Masucci and Schiefelbein 1994, Pitts et al. 1998) but
also have a strong effect on mutants that show morphological
characteristics very different from the hormone sensitivity
mutants ein2, etr1 and aux1. In similar experiments, Seifert et
al. (2004) have shown that the root hair mutant rhd1 is suppressed by exogenously applied ACC but not by IAA. This difference in hormone sensitivity of rhd1 and the der mutants
shows that ethylene and auxin do not act in the same way on
root hair development. Based on the phenotypes of the single
mutants of der8, der9/shv2, rhd1 and the corresponding double
1052
Arabidopsis der (deformed root hair) mutants
mutants with der1-2, these loci seem to function in different
processes during root hair development, indicating that the two
phytohormones are involved throughout the whole process of
root hair development. Ethylene and auxin were not able to
suppress the phenotype of the other der mutants tested. This
suggests that suppression of a mutant phenotype by the two
hormones depends on the function of the mutated gene rather
than on the developmental stage at which this gene is active.
Furthermore, the response to either only ethylene or ethylene
and auxin shows that the hormones have overlapping but not
identical functions in the processes leading to root hair formation. The suppression of the rhd1 mutant root hair phenotype
by ethylene is based on a change in the nucleotide sugar flux
caused by the suppression of two genes coding for UDP-glucose-4-epimerases. This change in sugar metabolism allows for
the accumulation of galactan-containing xyloglucan and arabinosylated galactan to wild-type levels, two cell wall polymers
that are strongly affected by the rhd1 mutation (Seifert et al.
2004). This demonstrates that ethylene can suppress mutants
that are affected in the composition of the cell wall. The analysis of the mutants der8 and der9/shv2 should give indications
as to whether ethylene also influences other cellular processes
during root hair development. If these two mutants also show
aberrant cell wall development, our results presented here
would indicate that auxin also has an influence on particular
steps of the cell wall formation process.
In conclusion, we have characterized a new series of
mutants affected in root hair development. The isolated der2–
der9 plants are affected in different phases of root hair development which are schematically indicated (Fig. 5). A subset of
the der mutants is responsive to ethylene and auxin, while
others, although functional in the same developmental phase,
are insensitive. Whether only mutants affected in particular
steps of cell wall formation are responsive to ethylene and
auxin or whether deficiencies in other steps also can be suppressed by the plant hormones remains to be investigated. The
map-based cloning of the der mutants and their molecular characterization will be a first step towards the identification of
their precise function and should also shed light on the mechanisms of hormone function during the process of growth and
development of this cell type.
medium and 0.6% phytagel (Sigma, Buchs, Switzerland), and vertically grown for 5 d at 22°C with a 16 h/8 h light/dark cycle. For propagation of plants, seedlings were transferred to soil and grown to
maturity under the same conditions.
For the double mutant analysis, crosses of the line der1-2 harboring a mis-sense mutation in the actin2 gene (Ringli et al. 2002) and the
different der mutants were propagated to the next generation. Among
segregating F2 plants, seedlings with the der2–der9 mutant phenotypes were selected from the corresponding population and analyzed
for the presence of the der1-2 mutation. Individuals heterozygous at
the der1-2 locus were identified and selected. In the subsequent F3
generation, the plants segregated only for the der1-2 mutation while
being homozygous mutant for the second der mutation. Due to the particular phenotype, double der1-2 mutant plants could be easily identified.
Mapping of the der loci
For mapping, the der mutants were crossed with wild-type
Arabidopsis of the ecotypes Columbia (Col) and Landsberg erecta
(Ler). Fifty mutant seedlings of the segregating F2 populations were
selected for mapping, using simple sequence length polymorphism
(SSLP) and cleaved amplified polymorphic sequence (CAPS) markers.
Microscopic analysis and root hair length measurement
The vertically grown seedlings were analyzed under a Leica stereomicroscope LZ M125. Differential interference contrast microscopy
was done with an axioplan microscope (Zeiss, Jena, Germany). For
scanning electron microscopy, seedlings grown on MS medium were
frozen in liquid propane and partly freeze-dried. After sputter coating
with platinum in a preparation chamber SCU 020 (BAL-TEC, Balzers, Liechtenstein), microscopic observations were performed in an
SEM 515 scanning electron microscope (Philips, The Netherlands).
For root hair length measurements, root hairs were photographed by
differential interference contrast microscopy, pictures printed and the
root hairs measured. For reference, a slide with a 10 µm scale was
used.
Hormone treatment
Seedlings were grown as described above for 4 d and then transferred to half-strength MS medium supplemented with 5×10–8 IAA,
5×10–7 ACC, or deionized water as a control, respectively, and vertically grown for 4 d prior to microscopic analysis by the stereomicroscope and measurement of the root hair length. For the measurement
of root hair length, the roots of seedlings were put on a glass microscopic slide under a coverslip and pictures were taken on the axioplan
microscope. The pictures were printed on paper and the length of the
root hairs was measured on the pictures and converted to µm using a
picture of a µm ruler that was taken in the same way.
Acknowledgments
Materials and Methods
Plant material and growth conditions
Seeds of Arabidopsis thaliana ecotype C24 were EMS mutagenized by Lehle Seeds (Round Rock, TX, USA), grown in M1 pools
of 1,000 plants of which the M2 seeds were collected. A total of 70,000
M2 seeds were screened for a mutant root hair phenotype. Selected
mutants were backcrossed three times with wild-type C24. The rhd1
and rhd2 mutants were obtained from the Arabidopsis Biological
Resource Center. For growth of seedlings, seeds were surface sterilized for 10 min in 1% (w/v) Na-hypochlorite, 0.03% (v/v) Triton X100, washed three times with sterile water and stratified for 3 d at 4°C.
Subsequently, the seeds were plated on sterile half-strength MS
We would like to thank Dr. Beat Frey for technical assistance
with scanning electron microscopy, and Dr. John Schiefelbein for providing the rhd6 mutant. This work was supported by the Swiss
National Science Foundation Grants no. 31–61419.00 and 3100A0103891.
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(Received March 1, 2005; Accepted April 21, 2005)