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Environmentally Induced Plasticity of Root Hair
Development in Arabidopsis1
Margarete Müller and Wolfgang Schmidt*
Institute of Biology, Humboldt University Berlin, Invalidenstrasse 42, 10115 Berlin, Germany
Postembryonic development of plants is dependent on both intrinsic genetic programs and environmental factors. The
plasticity of root hair patterning in response to environmental signals was investigated in the Columbia-0 wild type and 19
Arabidopsis mutants carrying lesions in various parts of the root hair developmental pathway by withholding phosphate
or iron (Fe) from the nutrient medium. In the aging primary root and in laterals of the wild type, the number of root hairs
increased in response to phosphate and Fe deficiency in a manner typical of each growth type. Although an increase in root
hair density in ⫺phosphorus plants was mainly achieved by the formation of extra hairs over both tangential and radial wall
of underlying cortical cells, roots of ⫺Fe plants were characterized by a high percentage of extra hairs with two tips. Root
hair patterning and hair length was differentially affected by the presence or absence of phosphate and Fe among the
genotypes under investigation, pointing to separate cascades of gene activation under all three growth conditions.
Divergence in root hair patterning was most pronounced among mutants with defects in genes that affect the first stages of
differentiation, suggesting that nutritional signals are perceived at an early stage of epidermal cell development. During
elongation of the root hairs, no differences in the requirement of gene products between the growth types were obvious. The
role of genes involved in root hair development in the aging primary root of Arabidopsis under the various growth
conditions is discussed.
1Root hairs are cylindrical, tubular structures perpendicular to the main cell axis derived from specialized epidermal cells, the trichoblasts. By greatly increasing the absorptive surface area, root hairs serve
in water and nutrient uptake, anchoring the plant to
the substrate, and are the site of interaction with
nitrogen-fixing bacteria. Root hairs are widespread
among vascular plants such as ferns, gymnosperms,
and angiosperms, underlining their importance in
plant/rhizosphere exchange processes. Similar to
pollen tubes, root hairs are tip-growing structures
formed by reorientation of cell extension. By maintaining a single growing point, new membrane is
continuously added at the tip by secretory vesicle
fusion. In Arabidopsis, root hairs are formed in a
predictable, position-dependent pattern. Root hairs
develop on the apical end of cells that overlie the
clefs of underlying cortical cells, and non-hair cells
are located over periclinal cortical walls. A number of
genes that control epidermal cell patterning and root
hair development have been defined by molecular
genetic studies (Schiefelbein, 2000, 2003; Dolan,
2001b; Grierson et al., 2001). Epistasis analysis has
shown that these genes do not act in a linear cascade
but in a complex and not strictly hierarchical pathway. Cell fate specification is regulated by a transcription factor cascade early in the meristem, includ1
This work was supported by the Deutsche Forschungsgemeinschaft.
* Corresponding author; email [email protected];
fax 49-30-20938725.
Article, publication date, and citation information can be found at
http://www.plantphysiol.org/cgi/doi/10.1104/pp. 103.029066.
ing the GL2 (GLABRA2) homeobox and the
WEREWOLF MYB-type transcription factor, which
are required for the non-hair fate. Another Arabidopsis gene, CPC (CAPRICE), encodes an MYB transcription factor mandatory for the hair fate (Wada et al.,
1997, 2002). GL2 gene expression is regulated by the
WD40 domain protein TTG (TRANSPARENT TESTA
GLABRA) by activating an R-like bHLH protein
(Scheres, 2000; Schiefelbein, 2000; Larkin et al., 2003).
Expression of WER has been shown to be biased by
positional cues and to regulate CPC and GL2 by
transcriptional feedback loops (Lee and Schiefelbein,
2002). CPC and TRY (TRYPTYCHON) act redundantly in lateral inhibition of root hair patterning
(Schellmann et al., 2002). Other specification genes
with less well-defined functions have been identified
by analysis of mutants harboring defects in root hair
patterning. The recessive mutants erh1 (ECTOPIC
ROOT HAIR1), pom1 (POM-POM1), and erh3 (ECTOPIC ROOT HAIR3) form additional root hairs in N
positions normally occupied by non-hair cells
(Schneider et al., 1997). ERH3 has been shown recently to encode a katanin-p60 protein (Webb et al.,
2003). The rhl (ROOT HAIRLESS) mutants rhl1, rhl2,
and rhl3 show disrupted differentiation of epidermal
cells in the meristem and form very few root hairs
(Schneider et al., 1997). Of the latter genes, only RHL1
has been cloned and was shown to encode a novel
protein localized in the nucleus.
After initiation, hairs begin to elongate by rapid
polarized expansion. During tip growth, biosynthesis
of new wall material, localized wall loosening, and
polarized targeting of vesicles from the endomem-
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Müller and Schmidt
brane system to the growing tip are regulated by
localized ion influxes and by the cytoskeleton (Ryan
et al., 2001). A number of mutants with altered root
hair phenotypes such as RHD (ROOT HAIR DEFECTIVE), TIP GROWTH1, COW1 (CAN OF WORMS1),
and KJK (KOJAK) have been isolated, but only in a
few cases is the underlying mechanism understood at
the molecular level (Schiefelbein, 2000; Grierson et
al., 2001; Ryan et al., 2001). In addition, ethylene and
auxin have been shown to affect patterning and initiation of root hairs, by acting either independently
from or downstream of the WER/TTG/CPC/GL2
pathway (Masucci and Schiefelbein, 1996). Root hairs
of mutants defective in ethylene and auxin signal
transduction form shorter hairs than the wild type or
no hairs at all, indicating that the ethylene and/or
auxin signal transduction pathway is required for
root hair elongation (Pitts et al., 1998; Dolan, 2001a).
Blocking the endogenous ethylene pathway was
shown, however, to be ineffective in affecting root
hair formation (Cho and Cosgrove, 2002).
The developmental program of roots is greatly influenced by environmental factors and can be modified according to the prevailing conditions. In particular, the availability of essential nutrients can affect
root development to allow for an increased uptake of
these nutrients (trophomorphogenesis; Forde and
Lorenzo, 2001; López-Bucio et al., 2003). These responses include alterations in growth rates, root
growth angle, and architecture of the root system.
Soil-immobile nutrients such as phosphate and iron
(Fe) can induce an increase in the root surface by an
increase in the density of root hairs when the supply
is inadequate to meet the demand of the plant (Gilroy
and Jones, 2000; Schmidt et al., 2000; Ma et al., 2001).
The acquisition of both Fe and phosphate depends on
a concerted action of an array of physiological and
morphological adaptations (Raghothama, 1999;
Schmidt, 1999, 2003; Curie and Briat, 2003; Hell and
Stephan, 2003). Although the sensors and signal
transduction mechanisms have not been identified so
far, it appears that the induction of trophomorphogenetic and physiological responses to Fe and phosphorus (P) starvation run parallel but separate
courses. Under both conditions, homeostatic compensation of the nutritional levels is systemically regulated, whereas morphological acclimations such as
transfer cell formation and the development of extra
root hairs are controlled by local signals (Raghothama, 2000; Schikora and Schmidt, 2001). This
implies that external phosphate and Fe levels can
be perceived by individual cells or tissues independent of the internal nutrient status. At which stage
of root hair development nutrient supply modulates
the fine-tuning of meristematic prepattern is
unclear.
This study is an attempt to elucidate how environmental signals modify cell differentiation in the root
epidermis of Arabidopsis. By analyzing mutants de410
fective in different processes of root hair development, we show that root hair patterning, initiation,
and elongation are variable under different growth
conditions to adapt to changes in the availability or
distribution of nutrients in the environment.
RESULTS
Wild-Type Responses
In contrast to seedling roots, in which almost all
epidermal cells that lie over the clefs of underlying
cortical cells (H position) form root hairs, in the aging
primary root and in laterals, the number of root hair
files is considerably reduced (Table I). In cross sections of primary roots, only three epidermal cells in
the H position develop into root hairs, resulting in
37% of the epidermal cells in this position being
differentiated as root hairs. As previously reported,
the formation of root hairs in Arabidopsis was sensitive to the availability of Fe and P in the growth
medium (Schmidt and Schikora, 2001). Under the
present conditions, the root hair frequency in the H
position in the primary root of the Columbia-0 wild
type was increased to 42% and 61% under Fe- and
P-deficient conditions, respectively (Table I; Fig. 1,
a–g). Hairs of P-deficient plants were markedly
longer than those formed in response to Fe starvation
or under control conditions; their length increased
from 0.2 ⫾ 0.01 up to 0.6 ⫾ 0.02 mm (Fig. 1, a–c). The
increase in root hair frequency in P-deficient plants is
associated with the development of hairs in N positions normally occupied by non-hair cells (Table I;
Fig. 1d). The formation of ectopic hairs was less
pronounced in roots of ⫺Fe plants (5.8% of the epidermal cells in the N position in ⫺P plants versus
2.2% in ⫺Fe plants). In Fe-deficient plants, about
one-third of the hairs were branched (Table I; Fig. 1,
h–j). Under consideration of the epidermal cell length
(control, 121.9 ⫾ 4.3 ␮m; ⫺Fe, 113.3 ⫾ 6.3 ␮m; and
⫺P, 118.8 ⫾ 2.1 ␮m), the number of root hairs per
millimeter was 25, 34.4, and 52.2 in control, ⫺Fe, and
⫺P plants, respectively. Based on the number of tips,
the frequency of hairs in ⫺Fe plants is 45.9 per millimeter of root length. Thus, it appears that the increase in absorptive surface is realized by different
developmental programs in ⫺Fe and ⫺P plants, either by the formation of branched hairs, as in the case
of Fe-deficient plants, or by the development of hairs
in both normal and ectopic positions and root hair
elongation, as in the case of P-deficient plants.
In lateral roots, the number of epidermal cells was
significantly lower under all growth conditions. The
increase in the number of root hairs in response to Fe
and P deficiency was similar to that of the primary
roots, with the exception of the number of hairs in the
N position in laterals of P-deficient plants, which was
markedly reduced.
Similar to roots of 3-week-old plants in which no
hairs during the first 3 d after transfer to Fe-depleted
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Plant Physiol. Vol. 134, 2004
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3.6 ⫾ 0.2
3.8 ⫾ 0.3
2.5 ⫾ 0.1
3.1 ⫾ 0.2
lrx
tip1-2
4.4 ⫾ 0.3
4.3 ⫾ 0.1a
3.4 ⫾ 0.1
3.3 ⫾ 0.2
rhd3
rhd4
5.1 ⫾ 0.3
6.3 ⫾ 0.4a
9.2 ⫾ 0.4a
5.3 ⫾ 0.3
1.2 ⫾ 0.3
0.2 ⫾ 0.1
0.9 ⫾ 0.1
1.1 ⫾ 0.2
0.6 ⫾ 0.1d
0.04 ⫾ 0.04 0.1 ⫾ 0.04
1.0 ⫾ 0.2
0.8 ⫾ 0.2
0.3 ⫾ 0.1a
1.7 ⫾ 0.2
0.2 ⫾ 0.1
5.8 ⫾ 0.2
a
0.06 ⫾ 0.04a
0a
0a
0a
0a
1.7 ⫾ 0.3
2.2 ⫾ 0.2a
0.06 ⫾ 0.03 0.9 ⫾ 0.3
0
0.09 ⫾ 0.03 0.7 ⫾ 0.2
0.02 ⫾ 0.02 0.3 ⫾ 0.2
0a
0a
0a
0a
0a
0a
1.0 ⫾ 0.2a
1.7 ⫾ 0.2a
5.1 ⫾ 0.3
5.5 ⫾ 0.3d
a
0.3 ⫾ 0.1
0.1 ⫾ 0.03 0a
2.8 ⫾ 0.8a
4.5 ⫾ 0.8a
4.6 ⫾ 0.6
0
0.6 ⫾ 0.1a
1.2 ⫾ 0.2
⫺P
0.08 ⫾ 0.03
0.4 ⫾ 0.3
0.06 ⫾ 0.04
0
2.8 ⫾ 0.2a
0.7 ⫾ 0.1a
0
0
0
0
0
0
0
0.06 ⫾ 0.03
0.05 ⫾ 0.03
0.02 ⫾ 0.02
0.04 ⫾ 0.04
0
0.1 ⫾ 0.1
0.07 ⫾ 0.04
0.1 ⫾ 0.1
0.8 ⫾ 0.2
1.3 ⫾ 0.3
1.0 ⫾ 0.3
0.08 ⫾ 0.07
0.04 ⫾ 0.03
0.04 ⫾ 0.03
0.04 ⫾ 0.03
4.1 ⫾ 0.3a
0.1 ⫾ 0.1a
0
3.5 ⫾ 0.4a
0.06 ⫾ 0.04
0
0
0
0
0
0
0.04 ⫾ 0.04
0.02 ⫾ 0.02
0
0
0
0
0
0
⫺P
3.1 ⫾ 0.4a
0a
0a
0a
0a
0a
0a
0.1 ⫾ 0.1a
0.5 ⫾ 0.1a
0.3 ⫾ 0.1a
0.3 ⫾ 0.2a
0.1 ⫾ 0.1a
1.3 ⫾ 0.5
0
0.8 ⫾ 0.2
1.3 ⫾ 0.2
⫺Fe
Branched Root Hairs
Control
a
28.5 ⫾ 0.5
25.3 ⫾ 0.8
27.3 ⫾ 0.5
28.8 ⫾ 0.7
33.0 ⫾ 0.7a
26.7 ⫾ 0.6
27.0 ⫾ 0.5
n.d.
31.2 ⫾ 0.5
n.d.
n.d.
n.d.c
28.3 ⫾ 0.5
32.3 ⫾ 0.5a
26.6 ⫾ 0.4
30.8 ⫾ 0.6a
29.4 ⫾ 0.5
27.3 ⫾ 1.7
23.6 ⫾ 0.4
25.7 ⫾ 0.3
25.4 ⫾ 0.2
26.9 ⫾ 0.4
30.2 ⫾ 0.5a
26.6 ⫾ 0.4
26.0 ⫾ 0.5
25.2 ⫾ 0.3
n.d.
30.7 ⫾ 0.9a
n.d.
n.d.
n.d.
26.5 ⫾ 0.9
31.0 ⫾ 0.4a
25.8 ⫾ 0.4
27.9 ⫾ 0.9
28.1 ⫾ 0.8
26.9 ⫾ 0.5
22.5 ⫾ 0.4
21.7 ⫾ 0.5a
26.3 ⫾ 0.3
⫺Fe
⫺P
27.6 ⫾ 0.6
25.8 ⫾ 0.6
26.9 ⫾ 0.3
30.3 ⫾ 0.2a
31.7 ⫾ 0.6a
28.0 ⫾ 0.5
28.6 ⫾ 0.3
26.1 ⫾ 0.7
30.4 ⫾ 0.5a
n.d.
n.d.
n.d.
27.9 ⫾ 0.4
30.7 ⫾ 0.8
27.9 ⫾ 0.6
30.0 ⫾ 0.4
30.8 ⫾ 0.6a
27.9 ⫾ 0.6
22.8 ⫾ 0.6
24.7 ⫾ 1a
28.8 ⫾ 0.6
Epidermal Cell No.
22.9 ⫾ 0.5a
27.7 ⫾ 0.6
Control
8.6 ⫾ 0.2
9.1 ⫾ 0.4
8.5 ⫾ 0.3
8.4 ⫾ 0.2
9.6 ⫾ 0.3a
8.1 ⫾ 0.1
8.6 ⫾ 0.4
n.d.
8.2 ⫾ 0.3
n.d.
n.d.
n.d.
9.5 ⫾ 0.4a
8.3 ⫾ 0.2
8.4 ⫾ 0.2
9.1 ⫾ 0.3
8.8 ⫾ 0.2
8.5 ⫾ 0.2
8.0 ⫾ 0
8.0 ⫾ 0
n.d.
8.4 ⫾ 0.2
n.d.
n.d.
n.d.
9.9 ⫾ 0.4a
9.6 ⫾ 0.3
8.4 ⫾ 0.2
8.5 ⫾ 0.2
8.9 ⫾ 0.3
8.2 ⫾ 0.2
8.2 ⫾ 0.1
8.3 ⫾ 0.3
8.4 ⫾ 0.3
⫺Fe
⫺P
8.3 ⫾ 0.1
8.6 ⫾ 0.3
9.0 ⫾ 0.3
8.5 ⫾ 0.2
9.9 ⫾ 0.2a
8.0 ⫾ 0
8.7 ⫾ 0.2
8.7 ⫾ 0.3
8.4 ⫾ 0.2
n.d.
n.d.
n.d.
9.9 ⫾ 0.3a
9.1 ⫾ 0.3a
8.3 ⫾ 0.1
8.7 ⫾ 0.3
8.9 ⫾ 0.2
8.4 ⫾ 0.2
8.3 ⫾ 0.2
8.8 ⫾ 0.2
8.2 ⫾ 0.1
Cortical Cell No.
9.4 ⫾ 0.3a
8.6 ⫾ 0.2
8.7 ⫾ 0.2
8.6 ⫾ 0.2
8.4 ⫾ 0.4
8.3 ⫾ 0.1
9.4 ⫾ 0.4
8.1 ⫾ 0.1
Control
a
b
Comparison with primary roots of the wild type under the respective growth condition results in a P value ⬍ 0.01 (in addition, these data are underlined).
Only small bulges appear
c
d
that were not visible in the cross sections. The quoted no. represents elongated root hairs.
n.d., Not determined.
The rhd2 mutant develop also small bulges, but in the cross
sections, distinct rhizodermal cells showed strong toluidine blue staining. These cells were counted as root hair cells.
2.9 ⫾ 0.6
4.5 ⫾ 0.3
0.6 ⫾ 0.2a
2.2 ⫾ 0.3
kjk
rhd2
5.5 ⫾ 0.3
4.0 ⫾ 0.3a
0
0
4.5 ⫾ 0.3
0.2 ⫾ 0.1a,b
trh1
0.07 ⫾ 0.05a
0a
rhd1
0a
0.04 ⫾ 0.04a,b
0a
0a
rhd6
0a
0a
0a
0a
0a
0a
rhl3
0a
0a
0
0a
0.8 ⫾ 0.1a
0.6 ⫾ 0.1a
0
4.1 ⫾ 0.5a
3.2 ⫾ 0.5a
0
0
0.02 ⫾ 0.02 0.2 ⫾ 0.1
0.4 ⫾ 0.1
⫺Fe
Root Hairs in N Position
Control
0.1 ⫾ 0.03
0a
0a
0a
4.8 ⫾ 0.3a
4.3 ⫾ 0.2a
erh3
0a
5.2 ⫾ 0.3
4.6 ⫾ 0.2a
3.7 ⫾ 0.2
erh1
rhl1
2.9 ⫾ 0.2a
2.6 ⫾ 0.2a
0.6 ⫾ 0.1a
cpc
rhl2
5.8 ⫾ 0.2a
4.6 ⫾ 0.4
5.2 ⫾ 0.3a
gl2
5.9 ⫾ 0.2a
5.6 ⫾ 0.3
3.9 ⫾ 0.3
4.2 ⫾ 0.4
3.5 ⫾ 0.5
3.8 ⫾ 0.2a
7.9 ⫾ 0.1
0.2 ⫾ 0.1a,b
0
5.1 ⫾ 0.1
5.0 ⫾ 0.2
⫺P
ttg1
7.8 ⫾ 0.2
3.8 ⫾ 0.2
3.5 ⫾ 0.1
3.0 ⫾ 0.2
2.6 ⫾ 0.2
⫺Fe
Root Hairs in H Position
Control
wer1
root
Seedling
col-0
root
Lateral
col-0
root
Primary
col-0
Gene
Table I. Effect of Fe and P deficiency on root hair formation in Arabidopsis wild-type and mutant plants
Values represent the no. (means ⫾ SE) of the indicated cell type per cell layer. Ten roots were scored for each genotype/treatment.
Plasticity of Root Hair Development in Arabidopsis
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411
Müller and Schmidt
Figure 1. Effects of Fe and P status on root hair
development of Arabidopsis wild-type roots. a,
Control. b, Fe-deficient root. c, P-deficient root.
d, Formation of ectopic root hairs (*) in roots of
P-deficient Arabidopsis plants. Cross sections of
a control root (e), a Fe-deficient root (f), and a
root grown in the absence of phosphate (g).
Cryo-scanning electron microscopies of a control root (h) and Fe-deficient roots (i and j).
Bar ⫽ 250 ␮m (a–c), 100 ␮m (d, h, and i), 50 ␮m
(j), and 20 ␮m (e–g).
conditions were formed (data not shown), 5-d-old
seedlings germinated on Fe-free medium displayed a
root-hairless phenotype (Table I). Thereafter, root
hairs developed in both cases. When seedlings were
germinated on P-deficient medium, the root hair patterning was not significantly different from control
conditions, although some hairs in the N position
were formed (Table I).
Cell Fate Determination and Initiation
To investigate whether defects in root hair development also affect the formation of hairs in response
to nutrient starvation, plants of 19 mutants harboring
defects in root hair development in the seedling stadium were grown on P- or Fe-free medium. Mutations in several genes have been shown to disrupt the
patterning of hair cells and non-hair cells in the meristematic zone of the roots or to affect the initiation of
the hairs. Defects in the TTG, GL2, and WER genes
result in roots producing hairs on almost all epidermal cells, and defects in the CPC gene cause the
formation of excess non-hair cells (Scheres, 2000).
WER encodes an MYB-related protein that is preferentially expressed in non-hair cells and has been
shown to represent an early regulator of epidermal
Figure 2. Roots of the wer and rhd1 mutants. a,
Bulge formation in roots of the wer mutant under control conditions. Rhizodermal development of rhd1 mutants in the primary root (b) and
in laterals (c). Bar ⫽ 100 ␮m.
412
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Plant Physiol. Vol. 134, 2004
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Plasticity of Root Hair Development in Arabidopsis
cell fate in the root and hypocotyl (Lee and Schiefelbein, 1999, 2002). The typical phenotype with nearly
all rhizodermal cells differentiated into root hairs
was only observed a few days after germination.
Thereafter, the aging primary root and newly formed
laterals formed only bulges under control conditions
that did not elongate (Fig. 2a). When grown in media
lacking either Fe or P, a high number of hairs were
formed in wer plants (Table I). The number of hairs in
the H position was nearly similar to that of the wild
type under Fe-deficient conditions and was slightly
reduced in P-deficient roots, but the frequency of
hairs in the N position was considerably enhanced
under both Fe and P deficiency (Table I). TTG encodes a small protein with WD40 repeats (Walker et
al., 1999). The reported root hair number of ttg seedlings ranges between 94 and 98 hairs per millimeter
of root length (Masucci and Schiefelbein, 1996; Wada
et al., 1997). When the number of hairs in the H
position is considered, the frequency of root hairs is
not significantly different from the wild type under
all three growth conditions (Table I). Similar to wer
roots, the frequency of hairs in the N position was
drastically enhanced in the absence of Fe and P. In
P-deficient roots, 29.9% of the epidermal cells in the
N position were developed into root hairs. In addition, roots of P-deficient ttg plants produced root
hairs that were clearly longer than those of the wild
type under similar conditions (1.0 ⫾ 0.02 mm, Fig.
3f). GL2 encodes a homeobox-containing putative
transcription factor and is preferentially expressed in
atrichoblasts (Masucci et al., 1996; Wada et al., 1997).
Root hair density of gl2 mutant plants was comparable with the ttg1 phenotype under the respective
conditions, although statistically significant higher
frequencies of hairs in the H positions were observed
under control and ⫺P conditions.
CPC encodes a protein with an MYB-like DNAbinding domain, negatively regulating the expression of GL2 and moving from the hairless cell to the
hair cell (Wada et al., 1997, 2002; Lee and Schiefelbein, 2002). Under control conditions, roots of cpc
mutants formed only a few hairs that were randomly
distributed along the root. The appearance of the
phenotypes of cpc roots under ⫺Fe and ⫺P conditions were similar to the wild type, although the
number of hairs was lower (P ⬍ 0.01; Fig. 3, a–c;
Table I). Ectopic root hairs were not produced in cpc
roots.
Despite marked changes in root hair frequency in
H and N positions among the wer, ttg, gl2, and cpc
mutants with respect to the wild type under all
growth conditions, the number of hairs was increased in response to P or Fe starvation. Thus, the
nutritional signal can be perceived and translated in
this group of mutants. Interestingly, in wer mutants,
branched root hairs with a number typical of ⫺Fe
wild-type plants were induced by Fe deficiency,
which stands in contrast to cpc, ttg, and gl2 plants in
Figure 3. Root tips of the cpc and ttg mutants
grown under control conditions or in the absence of Fe or P. a, cpc control. b, cpc ⫺Fe. c,
cpc ⫺P. d, ttg control. e, ttg ⫺Fe. f, ttg ⫺P.
Bar ⫽ 250 ␮m.
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413
Müller and Schmidt
which a significant lower number of branched hairs
was observed.
The RHL1 gene encodes a nuclear protein that is
required for root hair initiation (Schneider et al.,
1998). rhl mutants show no cytoplasmic differentiation of epidermal cells, indicating that the RHL1 gene
product is required for cell specification (Schneider et
al., 1998). Under the present conditions, rhl1 roots
remained hairless when grown on standard medium
or under either P or Fe deficiency, suggesting an
essential function of the RHL gene in the presence
and absence of Fe and phosphate (Table I). rhl2 and
rhl3 showed phenotypes similar to rhl1. All rhl mutants formed normal air-borne root hairs.
The ECTOPIC ROOT HAIR genes ERH1 and ERH3
are required for correct differentiation of rhizodermal cells. erh1 and erh3 mutants show a staining
pattern that is not strictly correlated with the position
of trichoblasts and atrichoblasts in the meristematic
region (Schneider et al., 1997). Both mutants are characterized by an increase in radial cell expansion, but
only in erh3 roots is the number of cortical cell files
irregular, ranging from seven to nine (Schneider et
al., 1997). Under all conditions, both mutants formed
more root hairs than wild-type plants in H positions
(Table I). This might in part be because of a significantly higher number of cortex cells in both mutants,
increasing the epidermal cell number in the H position. The difference between the mutants is not evident when only the hairs in ectopic positions are
considered, which are generally present in higher
frequency independent of the growth type. An increase in root hair frequency in response to P and Fe
deficiency was apparent, however, when compared
with the control. The number of branched root hairs
formed in response to Fe deficiency was significantly
lower in erh plants.
The RHD6 gene promotes root hair initiation (Masucci and Schiefelbein, 1994). In roots of the rhd6
mutant, no hairs were formed under all three growth
conditions, but the mutant formed normal hairs
when the roots were in contact with air.
Bulge Formation and Tip Growth
Defects in the RHD1 locus lead to larger bulges,
presumably because of disturbed control of cell wall
loosening (Schiefelbein and Somerville, 1990). Under
the present conditions, the primary root of rhd1
plants was completely devoid of hairs (Fig. 2b), with
the exception of ⫺P plants, in which some hairs were
observed occasionally (Table I). In laterals, root hair
density of rhd1 roots was not significantly different
from the wild type under all growth conditions,
when only those trichoblasts were considered that
succeeded in initiating root hairs. The non-hair cells
were characterized by wide bulges that comprised
the entire epidermal cell wall (Fig. 2c). Some laterals
displayed the wild-type phenotype. Of all mutants
414
under investigation, only in the rhd1 mutant were
clear differences in root hair density between the
primary and lateral roots observed (data not shown).
TRH1 is a potassium transporter that appears to be
crucial for tip growth under standard growth conditions but not for overall plant K⫹ nutrition because
the trh1 phenotype is not rescued by high external
potassium concentrations (Rigas et al., 2001; Desbrosses et al., 2003). Plants lacking the TRH1 gene
function formed only some bulges that did not elongate but did not form root hairs under control and
⫺Fe conditions. trh1 plants produced normal root
hairs under ⫺P conditions in a frequency and positional pattern that differed not considerably from the
wild type (Table I; Fig. 4, a–c). Thus, TRH1 is apparently not necessary for the formation of root hairs in
response to P deficiency.
LRX1 encodes a chimeric Leu-rich repeat/extensin
protein expressed in root hairs that is required for tip
growth to proceed properly (Baumberger et al.,
2001). lxr1 mutants show an irregular pattern of root
hair development with hairs often being shorter and
with swellings either at the basis or along the stalk.
Hairs of lrx1 roots were often ruptured at their tips. In
this mutant, fewer hairs than in the wild type were
formed in response to P deficiency. Under ⫺Fe conditions, most of the hairs (79.5%) in the lrx mutant
were branched, and a significant higher number of
branched hairs was also formed under control
conditions.
In tip1 roots, only very short root hairs were
formed, probably because of a defect in microtuble
dynamics (Ryan et al., 1998). The frequency of these
short root hairs did not differ markedly from the wild
type under all growth conditions (Table I; Fig. 4. d–f).
Hairs of ⫺P plants are longer than those of the control
plants. Under control and ⫺P conditions, but not in
medium lacking Fe, tip1 plants produce significantly
more cortical and epidermal cells. Independent of the
growth conditions, the majority of the hairs were
branched. Under control conditions, nearly all hairs
had two tips (87.5%), and the percentage of branched
hairs was slightly lower in ⫺Fe and ⫺P plants.
In contrast, mutation in the KJK (KOJAK/AtCSLD3)
gene causes a burst of the hairs after swelling formation. KJK was isolated by positional cloning and was
found to encode a cellulose synthase-like protein required during root hair elongation (Favery et al.,
2001). Grown under either Fe or P deficiency, kjk
mutants displayed a phenotype almost similar to the
wild type with respect to the root hair number, but
most of the hairs were ruptured at their tip and had
irregular lengths. Only under control conditions
were hairs in H positions formed in a considerably
lower frequency. In contrast to the wild type, only
very few branched root hairs developed in response
to Fe starvation. The same gene was cloned independently by Wang et al. (2001). In contrast to the kojak
mutation, bulges of csld3-1 mutants did not elongate.
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Copyright © 2004 American Society of Plant Biologists. All rights reserved.
Plasticity of Root Hair Development in Arabidopsis
Figure 4. Root tips of various mutants involved
in tip growth grown under control conditions or
in the absence of Fe or P. a, trh1 control. b, trh1
⫺Fe. c, trh1 ⫺P. d, tip1 control. e, tip1 ⫺Fe. f,
tip1 ⫺P. g, rhd3 control. h, rhd3 ⫺Fe. I, rhd3
⫺P. Bar ⫽ 250 ␮m.
The frequency of the bulges did not significantly
deviate from the wild type under all growth conditions (data not shown).
rhd2 mutants produced short root hairs under control and Fe-deficient conditions. When grown in
P-deficient medium, only bulges were produced that
did not elongate. The number of these bulges in ⫺P
plants was similar to the root hair frequency of the
wild type (Table I). RHD3 encodes a novel protein
with putative GTP-binding motifs, which is required
for regulated cell enlargement (Schiefelbein and
Somerville, 1990). rhd3 mutants produced also short
hairs under all conditions (Fig. 4. g–i). The number of
root hairs was similar to the wild type. RHD4 is a
further gene active in the maintenance of root hair
elongation. Root hair density under ⫺Fe and ⫺P
conditions in rhd4 mutants did not deviate significantly from the wild type except for the number of
hairs in H position under Fe deficiency, which was
significantly higher than in the wild type. Hairs of
the rhd4 mutant were generally shorter than in the
wild type. Interestingly, air-borne hairs of all mutants with defects in elongation resembled those of
the wild-type (data not shown).
DISCUSSION
Developmental/Environmental Regulation of Root
Hair Formation
The formation of extra root hairs in response to
suboptimal availability of Fe or phosphate are well-
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Plant Physiol. Vol. 134, 2004
Copyright © 2004 American Society of Plant Biologists. All rights reserved.
415
Müller and Schmidt
documented examples of morphologic acclimations
aiding in the uptake of limiting nutrients from the
soil (Forde and Lorenzo, 2001; Ma et al., 2001; LópezBucio et al., 2003). The number of root hair cells is
markedly reduced in the aging primary root when
compared with seedlings, in which all those cells
form root hairs that lie over the intercellular spaces of
underlying cortical cells (Schiefelbein, 2003; Table I).
This number is increased in response to Fe and P
deficiency, in a manner typical of each growth type.
In primary roots of P-deficient plants, 20% of the
total root hairs are in the N position, whereas in
laterals, only 10% of the hairs are ectopic. Because of
the fact that in 3-week-old plants the majority of the
root system consists of laterals, root hairs in N position are less important for the plant than the additional root hairs in H position. Ectopic root hairs in
laterals of ⫺Fe plants represent 5% of the total hairs;
thus, ectopic hairs appear to be less important under
Fe-deficient conditions.
Beside changes in root hair patterning, P-deficient
conditions evoked a significant increase in root hair
length (Figs. 1, 3, and 4), which is likely to improve P
acquisition. In seedling roots, all epidermal cells in
the H position developed into root hairs. When 5-dold plants were germinated on Fe- or P-free medium,
the roots showed a slight deviation from the control,
indicating that the environmental stimulus can be
perceived at an early developmental stage.
Mutants harboring defects in the WER/TTG/GL2
transcription factor cascade showed an increase in
root hair density in response to Fe and P deficiency
that resembled the wild type in most cases when the
hairs in H position are considered. This stands in
sharp contrast to the formation of ectopic hairs,
which are dramatically increased in this group of
mutants. Thus, it appears that in the aging primary
root, this group of genes is more important for the
fate of cells in N position than for adjusting the
number of hairs in normal position. An increase in
cortical cell number has been reported for P-deficient
Arabidopsis roots and was suggested to represent a
Figure 5. A model for the genetic control of root hair development
under ⫺Fe and ⫺P conditions in the aging primary root of Arabidopsis. In this model, those genes are mentioned whose rupturing
caused a phenotype significantly different from the wild type (P ⬍
0.01). Independent signal transduction cascades for the development of root hairs in response to Fe and P deficiency are hypothesized. Concerning cell fate specification, the WER/TTG/GL2 transcription factor cascade is important for the correct development of
non-hair cells in the N position under Fe and P deprivation. WER
and GL2 are also involved in the correct specification of rhizodermal cells in H position under P deficiency. CPC is required for the
differentiation of hair cells in both the H and N positions. ERH3 and
the RHL genes are crucial for the initiation of hairs under all
conditions. The initiation of root hairs is blocked in the absence of
functional RHD1 and RHD6 products independent of the nutrient
supply. During elongation of the root hairs, no differences in the
requirement of gene products between the growth types were obvious, with the exception of TRH1, which is not essential for the
formation of hairs in response to P deficiency. The development of
branched hairs is controlled by a separate cascade. For details, see
“Discussion.”
Table II. Phenotypes of the mutant plants under investigation
416
Gene
Mutant phenotype
Reference
wer1
ttg1
gl2
Cpc
erh1
erh3
rhd6
trh1
Lrx
tip1-2
Kjk
rhd2
rhd3
rhd4
Excessive root hairs
Excessive root hairs
Excessive root hairs
Fewer root hairs
Excessive root hairs
Excessive root hairs
No root hairs
No root hairs
Swollen basis, few hairs
Excessive bulging
Root hairs burst
Short root hairs
Short root hairs
Short root hairs
Lee and Schiefelbein (1999)
Galway et al. (1994)
Masucci et al. (1996)
Wada et al. (1997)
Schneider et al. (1997)
Schneider et al. (1997)
Masucci and Schiefelbein (1994)
Rigas et al. (2001)
Baumberger et al. (2001)
Schiefelbein et al. (1993)
Favery et al. (2001)
Schiefelbein and Somerville (1990)
Schiefelbein and Somerville (1990)
Galway et al. (1999)
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Plant Physiol. Vol. 134, 2004
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Plasticity of Root Hair Development in Arabidopsis
means of increasing the number of root hairs by
providing a higher number of epidermal cells in the
H position (Ma et al., 2001). Under the present conditions, an increase in cortical cell number was not
observed in the primary roots or in laterals. A higher
number of cortex cells relative to the wild type was
formed in the two erh mutants. This can in part
explain the increased frequency of hairs in the H
position under P deficiency but not under control
and ⫺Fe conditions, where the increase in root hairs
is higher than the increase in epidermal cell files in
the H position.
In ⫺Fe plants, the increase in absorptive surface
area is mainly achieved by the formation of branched
root hairs. Among the mutants with defects in cell
specification genes, only wer shows this response,
indicating that the WER product is not necessary for
inducing the phenotype typical of Fe-deficient plants.
Additional branched root hairs were also observed in
mutants with defects in maintaining cell shape such
as tip1 and lrx mutants, suggesting that this response
is induced downstream of cell specification. This
might be similar to trichome formation, where
branching is controlled after cell specification by a
microtubule-mediated multiple change in cell polarity (Ilgenfritz et al., 2003). Hairs with multiple tips
were also described for the ethylene- and auxininsensitive mutant axr2 (Wilson et al., 1990), pointing
to a possible involvement of these hormones in their
development. cDNA microarray analysis with Fedeficient Arabidopsis plants has revealed that upregulation of genes involved in ethylene synthesis
and signal transduction during Fe deficiency peaked
together with the maximal density of root hairs,
pointing to a possible involvement of ethylene in the
induction of the ⫺Fe root hair phenotype (Thimm et
al., 2002). A further potential component in the signal
cascade leading to branched hairs is auxin. Of the
hormone-associated genes present in the same cDNA
array, the auxin group was found to be the one in
which the highest percentage of differentially expressed genes was observed.
The data presented above can be interpreted in
terms of separate pathways induced in Fe- and
P-deficient plants. This difference is most pronounced in the trh1 mutant, in which root hairs were
only formed under P deficiency.
Because all mutants harboring defects in root hair
formation develop normally elongated air-born root
hairs, it can be assumed that these root hairs have a
different derivation.
A Model of Root Hair Formation in the Aging
Primary Root
Based on the phenotypical analysis of the mutants
listed in Table II, a model that groups the known
components involved in controlling epidermal patterning in Arabidopsis roots to the different path-
ways can be considered (Fig. 5). Independent cascade
events are assumed to regulate the development
ofroot hairs in response to P and Fe starvation. Differences concerning the required gene products are
also evident concerning the formation of hairs in the
H and N positions and for branched hairs. Functional
products of WER and GL2 appear to be required in all
three pathways for the development of hairs in both
H positions and TTG for the formation of hairs in the
N position. An exception is represented by roots of
Fe-deficient plants, in which WER is not required for
hairs in normal position. Interestingly, in contrast to
all other genes involved in cell specification, the WER
product is also not needed for the formation of
branched hairs. CPC is required for the hair fate in H
and N position, and a nonfunctioning CPC product
completely inhibits the formation of ectopic hairs.
CPC accumulates predominantly in non-hair cell files
and acts together with TRY in lateral suppression of
GL2 expression (Schellmann et al., 2002). Functional
products of the ERH and RHL genes are required for
the initiation of root hairs in the presence and absence of Fe and phosphate. In all growth types, root
hair initiation is blocked by defects in RHD6.
Under control and ⫺Fe conditions, proper regulation of the cellular events during bulge formation
and tip growth is dependent on functional TRH1.
Although sequence analysis and heterologous expression of this gene has shown that the TRH1 protein functions as a potassium transporter (Rigas et al.,
2001), the physiological function of TRH1 is not entirely clear (Desbrosses et al., 2003). From the normal
root hair development in ⫺P trh1 mutants, it can be
inferred that functional TRH1 protein is not required
for tip growth of root hairs under ⫺P conditions.
Thus, we assume that TRH1 plays a regulatory role in
root hair formation.
Concerning elongation of the hairs, no differences
in the effect of mutations in the genes involved in this
process were obvious between control, ⫺Fe, or ⫺P
conditions. rhd2, rhd3, and rhd4 mutants produce
short hairs under all conditions, indicating that the
RHD genes are important under all three conditions.
TIP1, CSLD, KJK, and LRX are further genes required
for a proper control of the duration and rate of tip
growth. Hence, it appears that after cell fate has been
determined, similar components are involved in the
further development of the hairs under all growth
conditions. Defects in this group of genes also affect
the formation of branched hairs in ⫺Fe plants.
In conclusion, our data show that the capacity to
respond to environmental information is achieved in
the aging primary root after intrinsic and positional
cues have determined cell fate in a largely invariant
way. Different pathways are evidently involved in
installing the phenotype typically of ⫺Fe and ⫺P
plants. The isolation of genes involved in the trophomorphogenetic signal transduction cascade will help
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Plant Physiol. Vol. 134, 2004
Copyright © 2004 American Society of Plant Biologists. All rights reserved.
417
Müller and Schmidt
to elucidate the processes leading to the fine tuning
of epidermal cell differentiation.
1500C (Oxford Cryosystems Ltd, Oxford, United Kingdom), roots were
observed in an S-3200N scanning electron microscope (Hitachi, Tokyo).
Received June 22, 2003; returned for revision August 4, 2003; accepted
September 25, 2003.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
The rhd1, rhd2, rhd3, rhd4, and wer mutants were kindly provided by John
Schiefelbein (University of Michigan, Ann Arbor); the trh1 mutant was
obtained from Polydefkis Hatzopoulos (Agricultural University of Athens);
tip1-2 and kojak were obtained from Eoin Ryan (University College, Dublin);
csld3-1 was obtained from Mieke Van Lijsebettens (University of Gent,
Belgium); rhl1, rhl2-1, rhl3-1, erh1, and erh3 were obtained from Katharina
Schneider (Max-Planck Institut für Züchtungsforschung, Köln, Germany);
rhd6 was obtained from Hyung-Taeg Cho (Pennsylvania State University,
State College); and lrx1 was obtained from Nicolas Baumberger (University
of Zurich). The other genetic stocks were obtained from the Arabidopsis
Biological Resource Center (Ohio State University, Columbus). All mutants
have been described elsewhere (see Table II).
Plants were grown in a growth chamber on an agar medium as described
by Estelle and Somerville (1987). The seeds were surface sterilized by
immersing them in 5% (v/v) NaOCl for 5 min and 96% (v/v) ethanol for 7
min, followed by four rinses in sterile water. The medium was composed of:
5 mm KNO3, 2 mm MgSO4, 2 mm Ca(NO3)2, 2.5 mm KH2PO4, 70 ␮m H3BO3,
14 ␮m MnCl2, 1 ␮m ZnSO4, 0.5 ␮m CuSO4, 10 ␮m NaCl, 0.2 ␮m Na2MoO4,
and 40 ␮m FeEDTA, and solidified with 0.55% (w/v) agar. Suc (43 mm) and
4.7 mm MES were included, and the pH was adjusted to 5.5. Seeds were
placed onto petri dishes containing agar medium and kept for 3 d at 4°C in
the dark before the plates were transferred to a growth chamber and grown
at 21°C in continuous light (50 ␮mol m⫺2 s⫺1, TL lamps, Philips, Eindhoven,
The Netherlands). Eleven days after sowing, plants were transferred to fresh
agar medium (control plants), medium without P (⫺P plants) or without Fe,
and medium with 100 ␮m 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine sulfonate
(⫺Fe plants). The lower concentration of K because of the absence of
KH2PO4 in the ⫺P medium was corrected by the addition of KCl. Plants
were analyzed 9 d after replanting to the different growth conditions. To
prepare the seedling plants, seeds were sown on either control, ⫺Fe, or ⫺P
medium and exposed to light after a stratification for 1 d at 4°C in the dark.
The epidermal cell patterning was determined 5 d after sowing.
Microscopy
Root hair patterns were analyzed in cross sections of 10 primary root
apical segments per genotype and treatment. The apical first cm of the root
tip was excised, washed in 0.5 ␮m CaSO4, and fixed in 3% (w/v) agarose
solution. Hand-cut sections from the root hair zone were stained with
toluidine blue, and one cell layer each was analyzed using an Eclipse E600
microscope (Nikon, Tokyo). The number of cortical and epidermal cells and
the rate of root hairs in the H and N positions and branched root hairs were
counted in five sections per root segment. If not otherwise marked, an
epidermal cell was scored as a root hair cell, if any protrusion was visible,
regardless of its length. Epidermal cell patterning of the seedling roots was
analyzed on the basis of two cross sections per root between the beginning
of the root hair zone and the collet region. In csld mutants, root hair
frequency was estimated under the stereomicroscope (see below) by counting the bulges in apical segments of 20 roots in the 2nd mm behind the apex.
The data were compared with the root hair number of the wild type
calculated by the same way. Statistical significance of differences between
mean values was determined using Student’s t test. Therefore, every mutant
was compared with the wild type under the respective growth condition.
To quantify the epidermal cell length in the root hair zone, dark-field
stereomicroscopy photographs of 10 root tips per genotype and treatment
were taken, and five cells per root tip were measured using Adobe Photoshop software (Adobe Systems, Mountain View, CA). Micrographs were
recorded with a Nikon Coolpix 990 digital camera, and dark-field stereomicroscopy was performed with a Stemi 2000-CS stereomicroscope (Zeiss,
Jena, Germany).
For cryo-scanning electron microscopy, root tips were fixed on the probe
plate with carbon adhesive and frozen in liquid nitrogen at ⫺175°C. After
sublimation at ⫺95°C in an Oxford-Cryochamber-/Transfersystem CTS
418
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