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The Type B Phosphatidylinositol-4-Phosphate 5-Kinase 3 IsEssential for Root Hair Formation in Arabidopsis thaliana W
Irene Stenzel,a Till Ischebeck,a Sabine Konig,a Anna Ho1ubowska,a Marta Sporysz,a
Bettina Hause,b and Ingo Heilmanna,1
a Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University Gottingen, 37077
Gottingen, Germanyb Department of Secondary Metabolism, Leibniz Institute for Plant Biochemistry, 06120 Halle (Saale), Germany
Root hairs are extensions of root epidermal cells and a model system for directional tip growth of plant cells. A previously
uncharacterized Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase gene (PIP5K3) was identified and found to
be expressed in the root cortex, epidermal cells, and root hairs. Recombinant PIP5K3 protein was catalytically active and
converted phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Arabidopsis mutant
plants homozygous for T-DNA–disrupted PIP5K3 alleles were compromised in root hair formation, a phenotype complemented
by expression of wild-type PIP5K3 cDNA under the control of a 1500-bp PIP5K3 promoter fragment. Root hair–specific PIP5K3
overexpression resulted in root hair deformation and loss of cell polarity with increasing accumulation of PIP5K3 transcript.
Using reestablishment of root hair formation in T-DNA mutants as a bioassay for physiological functionality of engineered
PIP5K3 variants, catalytic activity was found to be essential for physiological function, indicating that PtdIns(4,5)P2 formation is
required for root hair development. An N-terminal domain containing membrane occupation and recognition nexus repeats,
which is not required for catalytic activity, was found to be essential for the establishment of root hair growth. Fluorescence-
tagged PIP5K3 localized to the periphery of the apical region of root hair cells, possibly associating with the plasma membrane
and/or exocytotic vesicles. Transient heterologous expression of full-length PIP5K3 in tobacco (Nicotiana tabacum) pollen
tubes increased plasma membrane association of a PtdIns(4,5)P2-specific reporter in these tip-growing cells. The data
demonstrate that root hair development requires PIP5K3-dependent PtdIns(4,5)P2 production in the apical region of root hair
cells.
INTRODUCTION
The uptake of nutrients from the soil into plant roots is facilitated
by the formation of root hairs maximizing the root surface area.
Root hair formation is enhanced by nutrient-limiting conditions,
for instance, in response to limiting iron or inorganic phosphate
(Raghothama, 1999; Gilroy and Jones, 2000; Muller and
Schmidt, 2004), and may enable plants to colonize otherwise
nutrient-restrictive environments. Root hairs form as cytoplasmic
protrusions extending from root epidermal cells (Galway et al.,
1994) and, besides pollen tubes, are a model system for the
study of polar tip growth in plant cells.
The definition of sites of root hair initiation and genetic and cell
biological events driving hair cell elongation has been the focus
of previous studies (Schiefelbein, 2000; Pesch and Hulskamp,
2004; Samaj et al., 2004; Sieberer et al., 2005; Fischer et al.,
2006). The cellular machinery for polar tip growth of root hairs
involves the actin cytoskeleton (Voigt et al., 2005), regulatory
mitogen-activated protein kinases (Samaj et al., 2002), a variety
of regulatory GTP binding proteins (Preuss et al., 2004), and
factors required for vesicle trafficking (Yuen et al., 2005; Song
et al., 2006). Cytoskeletal structures, including microtubules,
help stabilize the elongated hair behind the actively growing tip
region and define the area where the plasma membrane is
expanded and cell wall material is deposited (Sieberer et al.,
2005).
To establish controlled polar tip growth, processes involved in
root hair elongation require tight spatial and temporal regulation.
Although it is not clear how the various elements of the complex
growth machinery are orchestrated, it has been proposed that
the polyphosphoinositide, phosphatidylinositol-4,5-bisphosphate
[PtdIns(4,5)P2], may play a role in the control of root hair growth
(Braun et al., 1999; Vincent et al., 2005). PtdIns(4,5)P2 can affect
multiple physiological processes in all eukaryotic cell types
studied so far by interacting with various protein partners that
are regulated in their biochemical activity or localization (Stevenson
et al., 2000; Mueller-Roeber and Pical, 2002; Meijer and Munnik,
2003; Balla, 2006). Examples for proteins regulated by PtdIns(4,5)P2
include plant ion channels and ATPases (Cote et al., 1996;
Suh and Hille, 2005), plant phospholipase D (Qin et al., 2002), the
actin-modifying enzymes profilin, cofilin, and gelsolin (Drobak
et al., 1994; Lemmon et al., 2002; Doughman et al., 2003;
Wasteneys and Galway, 2003; Wenk and De Camilli, 2004), and
mammalian SNARE complex proteins (Vicogne et al., 2006),
implying a role for PtdIns(4,5)P2 in vesicle fusion (Cremona and
1 Address correspondence to iheilma@uni-goettingen.de.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Irene Stenzel(istenze@uni-goettingen.de) and Ingo Heilmann (iheilma@uni-goettingen.de).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.107.052852
The Plant Cell, Vol. 20: 124–141, January 2008, www.plantcell.org ª 2008 American Society of Plant Biologists
De Camilli, 2001; Di Paolo et al., 2004; Gong et al., 2005;
Milosevic et al., 2005). PtdIns(4,5)P2 is also the substrate for
phospholipase C (PLC), yielding diacylglycerol and the soluble
second messenger, inositol 1,4,5-trisphosphate (Berridge, 1983;
Meijer and Munnik, 2003), required for Ca2þ release from internal
stores in both animal (Berridge, 1993) and plant cells (Alexandre
and Lassalles, 1990; Allen et al., 1995; Sanders et al., 2002).
Obviously, regulatory functions described for PtdIns(4,5)P2
include some of those required for a regulator of growth pro-
cesses in tip-growing cells. PtdIns(4,5)P2 has previously been
demonstrated to be involved in tip growth processes in pollen
tubes (Kost et al., 1999; Monteiro et al., 2005; Dowd et al., 2006),
a tip-growing system sharing some components of the growth
machinery with root hairs (Song et al., 2006). In pollen tubes,
Rac-type Rho proteins in the tip plasma membrane physically
associate with an enzyme activity producing PtdIns(4,5)P2 (Kost
et al., 1999), and PtdIns(4,5)P2 has been visualized in a plasma
membrane microdomain of the pollen tube tip (Kost et al., 1999).
As in pollen tubes, the presence of PtdIns(4,5)P2 in the plasma
membrane has previously been demonstrated for the root hair
cell tip (Braun et al., 1999), and phosphoinositides have been
postulated to contribute to the control of root hair development
(Braun et al., 1999; Vincent et al., 2005; Preuss et al., 2006). So
far, however, the enzymes and corresponding genes responsible
for generating PtdIns(4,5)P2 with regulatory function in pollen
tubes or root hairs have not been identified.
This study addresses the role of PtdIns(4,5)P2 formation as a
requirement for root hair growth. PtdIns(4,5)P2 is formed by
phosphorylation of its more abundant precursor lipid, phospha-
tidylinositol-4-phosphate (PtdIns4P), in the D-5 position of the
inositol ring, a reaction catalyzed by PtdIns4P 5-kinases (PI4P
5-kinases) (Drobak et al., 1999; Mueller-Roeber and Pical, 2002).
The Arabidopsis thaliana genome contains 11 genes with simi-
larity to animal PI4P 5-kinases (Mueller-Roeber and Pical, 2002),
only two of which have been experimentally characterized in
detail and been shown to encode enzymes with PI4P 5-kinase
activity in vitro (Elge et al., 2001; Perera et al., 2005). Plant PI4P
5-kinases, including amino acid sequences deduced from puta-
tive and uncharacterized genes, can be classified into types A
and B (Mueller-Roeber and Pical, 2002). The smaller type A
enzymes (isoforms 10 and 11) exhibit a domain structure similar
to that of animal and human PI4P 5-kinases, whereas type B
kinases (isoforms 1 to 9) are unique to plants in containing a large
additional N-terminal extension that includes several membrane
occupation and recognition nexus (MORN) repeats (Mueller-Roeber
and Pical, 2002). MORN repeats are found in various proteins of
both animal and plant origin that mediate protein membrane con-
tacts, such as the Arabidopsis ARC3 protein involved in plastidial
fission (Shimada et al., 2004; Maple et al., 2007) or junctophilins,
which mediate endomembrane-to-plasma membrane attach-
ment in mammalian cells (Takeshima et al., 2000). A recent
report indicates that the N terminus of Arabidopsis PI4P 5-kinase
isoform 1 (PIP5K1) has multiple regulatory effects on enzyme
activity and may guide subcellular localization of the enzyme
(Im et al., 2007). PI4P 5-kinases from different organisms have
been reported to associate with different subcellular locations,
including the plasma membrane of plants, yeast, and mammals
(Perera et al., 1999; Heilmann et al., 2001; Kobayashi et al., 2005;
Santarius et al., 2006), the nucleus of yeast and mammals
(Ciruela et al., 2000; Audhya and Emr, 2003; Santarius et al.,
2006), the actin cytoskeleton of plants, yeast, and mammals
(Desrivieres et al., 1998; Doughman et al., 2003; Davis et al.,
2007), and endomembranes of plants and mammals (Whatmore
et al., 1996; Heilmann et al., 1999; Im et al., 2007).
It has recently been reported that Arabidopsis plants carrying a
T-DNA insertion in the gene encoding the putative PI4P 5-kinase
isoform, PIP5K9, have elevated transcript levels for this gene,
correlated with overall reduced root length (Lou et al., 2007). No
enzymatic activity was demonstrated for a PIP5K9 gene product
in that study, and the restriction of the described phenotype to
roots has not been rationalized in light of the fact that the
disrupted gene is ubiquitously overexpressed throughout the
plant (Lou et al., 2007). Here, we show that a different member of
the Arabidopsis PI4P 5-kinase family, PIP5K3, is expressed in
root epidermal cells and root hairs and that recombinant PIP5K3
is catalytically active as a PI4P 5-kinase in vitro. Both under- and
overexpression of the PIP5K3 gene result in severe disturbance
of root hair directional growth and formation of short or morpho-
logically aberrant root hairs, respectively. The data presented
indicate that the formation of PtdIns(4,5)P2 by PIP5K3 in the
apical region of root hair cells is essential for polar tip growth.
RESULTS
PIP5K3 Is Expressed in Root Epidermis Cells and Root Hairs
In preparation of experiments to define possible physiological
roles of Arabidopsis PI4P 5-kinases, transcript array information
publicly accessible through the Genevestigator portal (Zimmermann
et al., 2004) was reviewed for the tissue-specific expression
patterns of all members of the Arabidopsis PI4P 5-kinase family
(Mueller-Roeber and Pical, 2002). Based on the Genevestigator
data available at the onset of this study, PIP5K3 was identified as
the only Arabidopsis PI4P 5-kinase gene with exclusive expres-
sion in roots, suggesting a specific function in root development
and making PIP5K3 the focus of this investigation. Direct exper-
imental verification of root-specific expression patterns of
PIP5K3 was achieved by RT-PCR (Figure 1A) and by expression
of fusions of a 1500-bp fragment of the 59-untranslated region
upstream of the PIP5K3-coding region with a b-glucuronidase
(GUS) reporter, followed by histochemical staining of transgenic
plant lines for GUS activity (Figures 1B to 1K). The respective
promoter GUS expression patterns were also tested for other
PI4P 5-kinase isoforms suggested by Genevestigator analysis to
be ubiquitously expressed, including in roots, and are described
and discussed in Supplemental Figure 1 and Supplemental Text
and References online). The promoter GUS experiments con-
firmed root-specific expression of the PIP5K3 gene (Figures 1B
and 1C) and revealed that the PIP5K3 promoter is active in cells
of the root cortex and epidermis (Figures 1D to 1F) and in root
hairs (Figure 1G). Only weak or no staining was detected in the
vascular tissue (Figure 1D), as evident especially in root cross
sections (Figures 1E and 1F). No GUS staining was observed with
the PIP5K3 promoter fragment in organs other than roots, as
indicated in Figures 1H to 1K.
PI4P 5-Kinase 3 and Root Hair Development 125
Arabidopsis PI4P 5-Kinase Isoforms Expressed in Roots
Are Active and Catalyze the Conversion of PtdIns4P
to PtdIns(4,5)P2
A prerequisite for further genetic and phenotypic studies is the
characterization of catalytic activity of the PIP5K3 protein or
other putative PI4P 5-kinase isoforms present in roots. Because
the PIP5K3 gene has not yet been functionally characterized,
PIP5K3 was heterologously expressed in Escherichia coli and
the recombinant protein tested for activity in vitro. The catalytic
activity of recombinant PIP5K3 and of some engineered PIP5K3
derivatives relevant to experiments described in Figures 3 to 5, 7,
Figure 1. PIP5K3 Is Expressed in Root Epidermis Cells and Root Hairs.
(A) Abundance of PIP5K3 transcript in different Arabidopsis organs according to RT-PCR analysis using a primer combination amplifying the full-length
PIP5K3 transcript. Flowers and siliques analyzed were taken from 6-week-old plants.
(B) to (K) Histochemical staining for GUS activity in different organs of transgenic plants expressing the GUSplus reporter gene under a 1500-bp
fragment of the PIP5K3 promoter. Samples were stained for GUS activity for 2 h ([C] to [E] and [G]) or for 24 h ([B], [F], and [H] to [K]). All plants shown
were analyzed at an age of 21 d, unless stated otherwise.
(B) Twenty-one-day-old plant.
(C) Two-day-old seedling.
(D) Root close-up. Bar ¼ 100 mm.
(E) and (F) Root cross sections. Bars ¼ 25 mm.
(G) Root hair close-up. Bar ¼ 100 mm.
(H) Flower bud.
(I) Open flower.
(J) Mature leaf.
(K) Stem and siliques. Inset: silique detail with developing seeds.
126 The Plant Cell
and 9 are given in Figure 2. Proteins derived from PIP5K3 that
were also tested (Figure 2) included a truncated PIP5K3 variant
lacking the N-terminal domains (DNT-MORN), a PIP5K3 deriva-
tive with mutated ATP binding site (K442A), a PIP5K3 derivative
mutated in three conserved positions of the catalytic site (S673A/
S680A/S682A; termed TripleA), and a truncated protein encoded
by the T-DNA–disrupted allele pip5k3-4 used for mutant studies
described below (pip5k3-4). Of the PIP5K3-derived proteins
tested, only full-length PIP5K3 and DNT-MORN had catalytic
activity, whereas the activity of the other PIP5K3 derivatives
tested was reduced to the limits of detection (Figure 2B). A minor
activity phosphorylating PtdIns3P to PtdIns(3,4)P2 was observed
for all full-length Arabidopsis PI4P 5-kinases tested, whereas no
activity was detected against PtdIns5P (Table 1). In addition, the
activities of root-expressed PI4P 5-kinase isoforms 1, 2, 7, 8, and
9 with the preferred substrate, PtdIns4P, were tested and are
given in Supplemental Figure 2 online. All proteins were recom-
binantly expressed as fusions to N-terminal maltose binding
protein (MBP) tags. Note that catalytic activity was not precluded
by the N-terminal addition of an MBP tag (Figure 2).
T-DNA Disruption of the PIP5K3 Gene Locus (At2g26420)
Reduces Root Hair Growth
To identify the physiological role of the PIP5K3 gene, a loss-of-
function approach was pursued. A search of the Salk Institute
Genomic Analysis Laboratory collection for T-DNA mutants of
the PIP5K3 gene locus (At2g26420) identified several indepen-
dent T-DNA insertions (Figure 3A) that were screened for homo-
zygosity. Homozygosity was inferred by the inability to amplify
the wild-type allele of PIP5K3 by PCR concomitant with positive
amplification of the T-DNA–tagged allele (see Supplemental
Figure 3 online). Insertion lines SALK_060590 and SALK_001546,
hereafter referred to as pip5k3-1 and pip5k3-2, carry T-DNA
insertions in the 59-untranslated region 248 and 48 bp upstream
of the PIP5K3-coding region, respectively. Insertion lines
SALK_000024 and SALK_026683 carry insertions in the third
intron or the sixth exon, respectively, and will be referred to as
pip5k3-3 and pip5k3-4 in the following text. The positions of the
T-DNA insertions were confirmed by sequencing of the corre-
sponding genomic loci. To test for altered PIP5K3 transcript
abundance in the T-DNA insertion lines, RNA gel blot experi-
ments were performed; however, PIP5K3 transcript levels were
too low to detect. The more sensitive RT-PCR analysis (Figure
3B) and quantitative real-time RT-PCR analysis (Figure 3C)
indicated that the insertion mutants pip5k3-2 and pip5k3-4 had
severely reduced PIP5K3 transcript levels compared with the wild
type, whereas only moderate transcript reduction was observed
in pip5k3-1 and pip5k3-3 plants. Homozygous pip5k3-4-plants
were chosen for a detailed characterization of the T-DNA–
disrupted transcript, and it was established that the correspond-
ing truncated mRNA encoded a truncated protein, pip5k3-4, that
lacks the extreme C terminus, including the activation loop
region (AL) involved in substrate binding (Kunz et al., 2000), and
was catalytically inactive after recombinant expression in E. coli
(Figure 2B).
Correlated to the reduction in PIP5K3 transcript levels (Figure
3C), all homozygous T-DNA insertion mutants exhibited reduced
root hair growth (Figure 4). Compared with wild-type plants
(Figure 4A), the reduction in root hair growth was most severe in
the lines pip5k3-2 (Figure 4C) and pip5k3-4 (Figure 4E) and less
Figure 2. Catalytic Activity of Arabidopsis PIP5K3 and Derived Proteins.
Proteins were recombinantly expressed in E. coli as fusions to N-terminal
MBP tags and were tested for their ability to convert PtdIns4P to
PtdIns(4,5)P2 in vitro.
(A) Schematic representation of PIP5K3 and derived proteins tested. NT,
N terminus (PIP5K31-49); MORN, MORN repeat domain (PIP5K350-227); Lin,
linker domain (PIP5K3228-307); Dim, dimerization domain (PIP5K3308-381);
Cat, catalytic domain (PIP5K3382-705). Arrowheads in K442A and TripleA
indicate mutagenized amino acid positions exchanging a catalytic Lys or
three conserved Ser residues in the catalytic domain for Ala residues,
respectively.
(B) Catalytic activity in recombinant E. coli extracts, as indicated.
Concentrations of recombinant proteins expressed in E. coli were
balanced according to protein gel blot analysis. Data represent the
means of three to four independent experiments 6 SD.
Table 1. Relative Activities of Arabidopsis PI4P 5-Kinase Isoforms
Expressed in Roots
Substrate PtdIns4P/PtdIns3P
Protein PtdIns3P PtdIns4P PtdIns5P Activity Ratio
MBP 2 6 0.3 4 6 0 – –
PIP5K1 5 6 0.8 205 6 17 – 41.0
PIP5K2 52 6 8 1206 6 156 – 23.0
PIP5K3 178 6 27 1972 6 66 – 11.0
PIP5K7 2 6 0.3 37 6 6 – 18.5
PIP5K8 3 6 0.5 27 6 3 – 9.0
PIP5K9 4 6 0.6 142 6 23 – 35.5
Catalytic activities were tested in vitro against different phosphatidy-
linositol-monophosphate substrates and indicate the rate of product for-
mation in fmol min�1. Recombinant protein concentrations were balanced
according to protein gel blot analysis. Data are the means of two inde-
pendent experiments 6 SD.
PI4P 5-Kinase 3 and Root Hair Development 127
pronounced in pip5k3-1 (Figure 4B) and pip5k3-3 (Figure 4D), as
summarized in Figure 4F.
Root Hair Development Is Reestablished in PIP5K3 T-DNA
Insertion Mutants Expressing Full-Length PIP5K3
The observation that the loss of PIP5K3-transcript was corre-
lated with reduced root hair growth (Figures 3 and 4) was
unexpected because the expression of five other active PI4P
5-kinase isoforms in roots (see Supplemental Figures 1 and 2
online) had suggested some functional redundancy. To verify
that the observed reduction in root hair growth was indeed due to
the disruption of the PIP5K3 gene, complementation of the
pip5k3-4 mutant phenotype (Figures 4E and 4F) was attempted
using full-length PIP5K3 or various PIP5K3 derivatives, as indi-
cated (Figure 5). The complementation constructs contained the
respective cDNA clones behind the 1500-bp PIP5K3 promoter
fragment used for the GUS expression experiments shown in
Figure 1 and encoded the respective proteins as fusions to
N-terminal enhanced yellow fluorescent protein (EYFP) tags.
Complementation was evaluated in comparison to nontrans-
formed wild-type plants (Figure 5A) and to pip5k3-4 mutants
(Figure 5B) grown in parallel. The genotypes of the plant lines
used and the presence of correctly sized transcripts correspond-
ing to the ectopically introduced PIP5K3 constructs were deter-
mined by PCR and RT-PCR, respectively (Figure 5G). Expression
of PIP5K3 in the pip5k3-4 mutant resulted in reestablishment of
normal root hair growth (Figure 5C), and root hairs were at an
average even longer than those of wild-type controls (Figure 5H).
Using the reestablishment of root hair growth in the pip5k3-4
mutant (cf. Figure 3) as a bioassay, the functionality of the
PIP5K3-derived proteins was tested. The truncated DNT-MORN
or the inactive K442A or TripleA proteins did not reestablish root
hair formation in the pip5k3-4 background (Figures 5D to 5F), as
summarized in Figure 5H. Root hair stubs forming with expres-
sion of K442A (Figure 5E) appeared thinner than those seen in
other transgenics. As the levels of the introduced proteins in root
hairs were too low to be detected by EYFP fluorescence or by
immunoblotting, expression of the transgenes was verified by
monitoring transcript levels for the various PIP5K3 derivatives
by quantitative real-time RT-PCR (Figure 5I), indicating that the
expression levels of the EYFP-tagged PIP5K3 transgenes intro-
duced into the pip5k3-4 mutant did not exceed the level of
PIP5K3 expression in wild-type plants by more than an average
of ;30%.
Aberrant Root Hair Growth and Morphology with
Overproduction of PIP5K3
The underexpression and complementation data for the PIP5K3
gene (Figures 4 and 5, respectively) indicated that PIP5K3 was an
essential regulator of root hair development and led us to ask
whether root hair formation would also be disturbed by overpro-
duction of EYFP-tagged PIP5K3. The stronger root hair–specific
promoter of Arabidopsis expansin 7 (EXP7) (Cho and Cosgrove,
2002) was used to drive ectopic overexpression of PIP5K3 in root
hairs of wild-type Arabidopsis plants. Compared with nontrans-
formed wild-type plants (Figure 6A), increasing degrees of over-
expression (Figures 6B to 6D) resulted in increasingly deformed
root hairs. From the highly polar appearance in wild-type plants
(Figure 6E) root hair morphology was altered with increasing
PIP5K3 overexpression toward thicker hairs (Figure 6F), curling
hairs (Figure 6G), and, with the highest degree of overexpression,
globular structures (Figure 6H), indicating a gradual loss of
cellular polarity. Increased PIP5K3 transcript levels in the over-
expressor lines were documented independently by quantitative
Figure 3. T-DNA Disruption of the PIP5K3 Gene Locus (At2g26420)
Results in Reduced PIP5K3 Transcript Levels.
(A) Structure of the genomic PIP5K3 locus with positions of the four
T-DNA insertions, pip5k3-1, pip5k3-2, pip5k3-3, and pip5k3-4. Arrows
indicate the position of primers used for RT-PCR analysis of transcript
levels.
(B) Abundance of full-length PIP5K3 transcript in roots of 3- to 4-week-
old plants homozygous for the respective T-DNA insertions, as deter-
mined by RT-PCR. Note that a pip5k3-4 protein would be truncated at the
extreme C terminus and catalytically inactive (Figure 2B). ACT8, control.
(C) Real-time RT-PCR analysis of PIP5K3 transcript levels in roots of
3- to 4-week-old wild-type or mutant plants, as indicated, using a primer
combination amplifying a 64-bp fragment of the cDNA stretch encoding
the catalytic region of the PIP5K3 protein. Dashed line, wild-type PIP5K3
transcript levels. Real-time RT-PCR data are given relative to transcript
levels detected in wild-type plants and represent the mean 6 SE of two
independent experiments.
128 The Plant Cell
real-time RT-PCR (Figure 6I) and by RT-PCR (Figure 6I, inset).
Data shown are from representative transgenic lines. Morpho-
logical changes as those reported were found to correlate with
transcript accumulation in eight of eight independent transgenic
lines.
Effects of Ectopic Expression of K442A and Truncated
DNT-MORN on Root Hair Morphology
Because under- and overexpression of PIP5K3 both affected
root hair morphology, the effects of overexpressing the inactive
K442A protein or the truncated DNT-MORN protein were tested.
Figure 4. Reduced Root Hair Growth of T-DNA Insertion Mutants of PIP5K3.
Plants were grown on agar plates for 8 d, and digital images were taken.
(A) to (E) Representative root hair phenotypes. Bars ¼ 100 mm.
(A) Wild-type plants.
(B) pip5k3-1 mutant.
(C) pip5k3-2 mutant.
(D) pip5k3-3 mutant.
(E) pip5k3-4 mutant.
(F) Quantification of root hair length in the wild type and in plants carrying T-DNA insertions, as indicated. The asterisks indicate significantly reduced
root hair length compared with wild-type controls according to a Student’s t test (*, P < 0.05; **, P < 0.01; n > 300).
PI4P 5-Kinase 3 and Root Hair Development 129
Figure 5. Root Hair Phenotypes of pip5k3-4 Plants Ectopically Expressing PIP5K3 or Derived Proteins.
Complementation of the pip5k3-4 mutant phenotype was tested by ectopic expression of PIP5K3 or various derived proteins under a 1500-bp fragment
of the intrinsic PIP5K3 promoter in the pip5k3-4 background.
(A) and (B) Root hair phenotypes of wild-type plants (A) and pip5k3-4 mutant plants (B) grown in parallel.
(C) to (F) Root hair phenotypes of pip5k3-4 plants with expression of PIP5K3 (C), DNT-MORN (PIPK3228-705; [D]); K442A (E), and TripleA (F). Bars¼ 200
mm. Images are representative for results obtained with at least five independent transgenic lines.
(G) The presence of the T-DNA insertions was shown by PCR using genomic DNA as a template and a combination of primers specific for the T-DNA
130 The Plant Cell
Both proteins were expressed as fusions to the fluorescence tag
RedStar. Root hairs of nontransformed wild-type plants (Figure
7A) and of plants expressing RedStar alone (Figure 7B) did not
differ in length. The introduction of RedStar-tagged K442A under
the EXP7 promoter resulted in root hair deformation and branch-
ing (Figure 7C). Overexpression of RedStar-tagged DNT-MORN
protein (Figure 7D) resulted in a phenotype similar to that ob-
served with K442A. Note that DNT-MORN was catalytically
active (Figure 2B) but lacks an N-terminal domain of unknown
physiological role. Phenotypes were observed in eight out of
eight (K442A) and six out of six (DNT-MORN) transgenic lines;
however, root hairs were never globally altered, and all plants
also had root areas with wild-type-like root hairs. The presence
or absence of K442A and DNT-MORN transcripts of the correct
sizes was verified by RT-PCR (Figure 7E). The PIP5K3 expres-
sion levels in the control lines and the overexpressors were
determined by quantitative real-time RT-PCR (Figure 7F) and
were found to be substantially increased over wild-type PIP5K3
transcript levels. Note that the real-time RT-PCR does not
distinguish between the wild-type allele and the mutated or
truncated variant of the PIP5K3 gene. The data are consistent
with dominant-negative effects resulting from overexpression of
K442A or DNT-MORN.
PIP5K3 Localizes to the Apical Region of Root Hair Cells
Because enzymes controlling cell polarity and polar growth
distribute to their subcellular sites of action, the subcellular
localization of PIP5K3 in root hairs was investigated by confocal
laser scanning microscopy. An EYFP tag was N-terminally linked
to PIP5K3, and the fusion protein was expressed under the EXP7
promoter in wild-type Arabidopsis plants. The resulting distribu-
tion of EYFP fluorescence is shown in Figure 8. Confocal images
of the bright-field (Figure 8A) and the EYFP channel (Figure 8B)
indicating PIP5K3 localization were synchronously recorded. A
merge of these images is given in Figure 8C. The distribution of
EYFP fluorescence indicates that the EYFP-PIP5K3 fusion pro-
tein is restricted to the periphery of the apical region of root hair
cells. In root hairs expressing EYFP-PIP5K3 under the control of
the EXP7 promoter, the EYFP-PIP5K3 fluorescence did not show
a tight distribution typical for plasma membrane proteins. While
formation of PtdIns(4,5)P2 was expected to occur at the plasma
membrane (Braun et al., 1999; Kost et al., 1999; Perera et al.,
1999; Heilmann et al., 2001; Kobayashi et al., 2005; van Leeuwen
et al., 2007), EYFP-PIP5K3 also decorated a conical pattern of
small cytosolic particles in the apex of root hair cells in addition to
possible plasma membrane association (Figure 8D). To docu-
ment localization of EYFP fluorescence in relation to root hair
autofluorescence, a confocal cross section of 1.6 mm thickness
close to the apical tip of a root hair was scanned, indicating a ring
of EYFP fluorescence immediately inside the autofluorescing cell
wall (Figure 8E). The distribution of EYFP-PIP5K3 fluorescence
reported was only observed in growing root hairs close to the
root tip, such as that shown in Figure 8, whereas older root hairs
exhibited only autofluorescence.
Expression of PIP5K3 Increases Plasma Membrane Levels
of PtdIns(4,5)P2
To determine whether plasma membrane PtdIns(4,5)P2 levels
were influenced by the expression of PIP5K3 in tip-growing plant
cells, PIP5K3 and K442A were each transiently expressed in
tobacco (Nicotiana tabacum) pollen tubes, together with the
RedStar-tagged Pleckstrin homology (PH) domain of the human
PLCd1 (HsPLCd1-PH domain), which specifically recognizes
PtdIns(4,5)P2 (Varnai and Balla, 1998) (Figure 9). Pollen tubes
were chosen instead of root hairs because the system is similar
to root hairs in many respects, it can easily be transiently trans-
formed by particle bombardment, and its autofluorescence is
negligible. The active and inactive proteins both localized to the
apical plasma membrane of the pollen tube tip in a pattern
resembling the distribution of EYFP-PIP5K3 in root hair cells (see
Supplemental Figure 4 online; compare Figures 8B and 8D). The
HsPLCd1-PH domain reporter (Figures 9A and 9B, middle pan-
els) also localized to the apical plasma membrane of the pollen
tubes, as previously reported (Kost et al., 1999; Dowd et al.,
2006). The right panel in Figure 9A illustrates roughly equal
intensities of PIP5K3 and HsPLCd1-PH domain, resulting in a
yellow color on the merged image, whereas unequal intensities of
K442A and HsPLCd1-PH domain shown in Figure 9B result in
green plasma membrane fluorescence of the merged image.
When fluorescence intensities were quantified (Figure 9C), the
fluorescence distribution of RedStar-HsPLCd1-PH with coex-
pression of active PIP5K3 was clearly plasma membrane associ-
ated (Figure 9C, top right panel), whereas equivalent expression of
the inactive K442A resulted in a more diffuse distribution of the
PtdIns(4,5)P2 reporter (Figure 9C, bottom right panel), indicating
that a higher proportion of the reporter remained in the cyto-
plasm. While synchronous imaging of PIP5K3-derived proteins
Figure 5. (continued).
insert (sense) and the PIP5K3 gene (antisense). The presence of correctly sized transcripts corresponding to the ectopic transgenes introduced was
tested by RT-PCR on root RNA, as indicated, using a primer combination specific for the cDNA stretch encoding the N-terminal EYFP tag (sense) and
for that encoding the extreme C terminus of the PIP5K3 protein (antisense). ACT8 served as an RNA loading control for the RT-PCR experiment. All
plants were analyzed 8 d after germination.
(H) Quantification of root hair lengths in 8-d-old wild type and transgenics. EYFP, control expressing EYFP alone under the PIP5K3 promoter fragment.
Asterisks indicate significantly increased root hair length compared with those of the pip5k3-4 background according to a Student’s t test (**, P < 0.01;
n > 300).
(I) Real-time RT-PCR analysis of PIP5K3 transcript levels in roots of wild-type, mutant, and complemented mutant plants, as indicated. The primer
combination used amplified a 64-bp fragment of the cDNA stretch encoding the catalytic region of the PIP5K3 protein. Real-time RT-PCR data are given
relative to transcript levels detected in wild-type plants and represent the mean 6 SE of two independent experiments.
PI4P 5-Kinase 3 and Root Hair Development 131
with the HsPLCd1-PH domain was successful in pollen tubes
(Figure 9), no interpretable images were obtained in correspond-
ing experiments on root hairs because of interfering autofluo-
rescence at the low HsPLCd1-PH expression levels required for
meaningful interpretation.
DISCUSSION
The data presented indicate an essential role for the PI4P
5-kinase PIP5K3 in the development of root hairs in Arabidopsis.
The requirement of PIP5K3 for root hair formation must be
reviewed in light of its catalytic activity and its capability to
generate PtdIns(4,5)P2 (Figure 2). The in vitro activity of recom-
binant PIP5K3 protein indicates a clear preference for the con-
version of PtdIns4P to PtdIns(4,5)P2 (Table 1), which is consistent
with the presence of a conserved Glu residue (Glu-668) in the AL
of PIP5K3 implicated in determining PI4P 5-kinase specificity
(Kunz et al., 2000). A low but detectable activity of PIP5K3 and
other isoforms against PtdIns3P (Table 1) is consistent with side
activities previously reported for PIP5K1 (Elge et al., 2001) and
PIP5K10 (Perera et al., 2005), which also exhibit minor activity
with this substrate. For the ubiquitous PIP5K1, enhanced activity
Figure 6. Aberrant Root Hair Morphology with Overproduction of PIP5K3.
The wild-type PIP5K3 allele was expressed under the root-hair-specific EXP7 promoter in wild-type plants.
(A) Root hair morphology of wild-type controls grown in parallel.
(B) to (D) Overexpressor lines shown were chosen to document morphological alteations with increasing levels of PIP5K3 transcript.
(E) to (H) Magnifications of root hairs exhibiting morphologies characteristic for wild-type plants (E) or for plants with increasing levels of PIP5K3
expression ([F] to [H]). Bars ¼ 200 mm in (A) to (D) and 100 mm (E) to (H).
(I) PIP5K3 transcript accumulation was monitored independently by real-time RT-PCR, using a primer combination amplifying a 64-bp fragment of
PIP5K3 as described above and by RT-PCR-analysis (inset) using a primer combination amplifying the full-length PIP5K3 transcript. Real-time RT-PCR
data are given relative to transcript levels detected in wild-type plants and represent the mean 6 SE of two independent experiments. PIP5K3, transcript
levels; ACT8, control. A to D refer to panels above. All plants were analyzed 8 d after germination. Altered root hair morphology correlated to PIP5K3
expression levels was observed in eight out of eight transgenic lines analyzed.
132 The Plant Cell
was reported with removal of the MORN domain of that protein,
indicating an autoinhibitory function for the MORN domain (Im
et al., 2007). In our in vitro activity tests with the PIP5K3-derived
DNT-MORN protein, no such enhancement of PI4P 5-kinase
activity was observed (Figure 2B). Possible explanations include
that autoinhibitory effects previously described (Im et al., 2007)
were specific for PIP5K1 or that in this study the MBP tag
N-terminally attached to the MORN domain of PIP5K3 interfered
with its regulatory functions. The latter scenario appears unlikely,
however, in light of the observation that in vivo functionality
of PIP5K3 was given with expression of PIP5K3 fused to an
N-terminal EYFP reporter (Figure 5C), which is roughly equal in
size and bulk to the MBP tag. The activity tests demonstrate the
importance of the extreme C-terminal end of the PIP5K3 protein,
including the AL, for catalytic activity because the truncated
pip5k3-4 protein was not active (Figure 2B). The loss of catalytic
activity with exchange of three conserved Ser residues in the
TripleA mutant (Figure 2B) attributes importance to the extreme
C-terminal portion of the PIP5K3 protein and suggests PIP5K3
positions Ser-673, Ser-680, and Ser-682 as candidate sites for
posttranslational regulation.
The results of in vitro biochemical characterization of PIP5K3
and its engineered variants (Figure 2) were in congruence with
evidence from genetic studies and complementation tests. In-
dependent T-DNA insertion mutants for the PIP5K3 gene, fore-
most those homozygous for alleles pip5k3-2 and pip5k3-4, were
compromised in root hair development (Figure 4), indicating that
the observed effects on root hair growth were due to altered
expression of PIP5K3, rather than to additional T-DNA insertions
elsewhere in the genome. This notion is further supported by the
fact that PIP5K3 T-DNA insertion mutants ectopically expressing
the wild-type allele of PIP5K3 under its intrinsic promoter ex-
hibited wild-type-like root hair growth (Figure 5C) and that
overexpression of the inactive K442A exerted subtle but repro-
ducible dominant-negative effects on root hair growth (Figure
7C). The observation that elimination of a single gene encoding
one of 11 PI4P 5-kinases (Figures 3 and 4), six of which are
expressed in roots (shown for PIP5K2 and PIP5K9 in Supple-
mental Figure 1 online) and are all catalytically active (Table 1;
see Supplemental Figure 2 online), nonetheless resulted in a
severe phenotype indicates that other PI4P 5-kinases, such as
PIP5K9 also expressed in root hairs (see Supplemental Figure
1 online), did not functionally compensate for the loss of PIP5K3
expression in regard to root hair development. This result is
consistent with results presented in Figure 2 and Supplemental
Figure 7. Effects of K442A or DNT-MORN Expression on Root Hair
Growth.
(A) to (D) The inactive PIP5K3-derived K442A protein or the truncated
DNT-MORN protein was expressed in wild-type plants as fusions to
N-terminal RedStar tags under the control of the EXP7 promoter. Images
were taken from 8-d-old plants; the transgenic lines are representative
for eight (K442A) and six (DNT-MORN) independent lines tested. To
varying degrees, all lines also exhibited regions with unaltered root hairs.
Bars ¼ 200 mm.
(A) Wild-type control.
(B) RedStar control.
(C) and (D) Root hair morphology observed with expression of K442A (C)
or DNT-MORN (D).
(E) RT-PCR detection of correctly sized ectopic PIP5K3 transcripts for
the constructs introduced, as indicated, using a primer combination
specific for the cDNA encoding the RedStar tag (sense) and for that of the
extreme C terminus of the PIP5K3-derived expressed proteins (anti-
sense). RedStar, RedStar transcript, as amplified with RedStar-specific
primers; ACT8, actin control.
(F) Real-time RT-PCR analysis of PIP5K3 transcript levels in plant lines,
as indicated, using a primer combination for the 64-bp PIP5K3 fragment
described above. Dashed line, wild-type PIP5K3 transcript level. Real-
time RT-PCR data are given relative to transcript levels detected in
wild-type plants and represent the mean 6 SE of two independent ex-
periments. Note that the real-time RT-PCR does not distinguish between
wild-type and mutated alleles of the PIP5K3 gene. All plants were
analyzed 8 d after germination.
PI4P 5-Kinase 3 and Root Hair Development 133
Figure 2 online, indicating that recombinant PIP5K3 is by far the
most catalytically active PI4P 5-kinase of the isoforms expressed
in roots. It is interesting to note that the pip5k3-1 mutant carrying
a T-DNA insertion only 248 bp upstream of the coding region was
only moderately impaired in PIP5K3 transcript accumulation
(Figure 3C) and also only moderately impaired in root hair growth
(Figure 4B), suggesting that the 248-bp region still in place may
contain most necessary regulatory elements of the PIP5K3
promoter. It was also a surprise to observe that the pip5k3-3
mutant exhibited substantial PIP5K3 transcript levels and that
the T-DNA insertion in intron three did not prevent efficient mRNA
splicing (Figure 3). From the phenotypes exhibited by the mutant
lines pip5k3-2 and pip5k3-4, which had strongly reduced PIP5K3
transcript levels (Figure 3C), it is clear that PIP5K3 has an
important function in the control of root hair growth.
To better understand the function of PIP5K3 in root hair
formation, inactive or truncated variants of PIP5K3 were intro-
duced into the pip5k3-4 mutant background (Figure 5), and the
capability of these constructs to reestablish root hair formation
was statistically evaluated (Figure 5H). While ectopic expression
of the wild-type allele of the PIP5K3 gene resulted in full com-
plementation of the pip5k3-4 phenotype (Figure 5C), catalytically
inactive K442A (Figure 5E) and TripleA (Figure 5F) did not
reestablish root hair formation in the pip5k3-4 background.
Whereas PIP5K3 protein levels in root hairs were too low to be
immunodetected, the following arguments indicate that the key
truncations used in this experiment, K442A and DNT-MORN,
were introduced into plants as functional proteins: (1) pheno-
types observed with expression of PIP5K3 and K442A contrasted
radically (cf. Figures 5C and 5E), although the proteins differed
only in one amino acid position not likely to result in a dramatic
difference in protein stability; (2) overexpression of DNT-MORN
under the EXP7 promoter in wild-type plants resulted in de-
formed root hairs (Figure 7D), indicating that the DNT-MORN
protein was functional in those plants and likely not instable. The
combined results indicate that catalytic activity and, thus,
PtdIns(4,5)P2 production are required for root hair formation.
The phenotype with TripleA expression also supports the notion
that a truncated pip5k3-4 transcript does not encode a functional
protein, which correlates well with the observed phenotype of the
pip5k3-4 mutant (Figure 4E). It is interesting to note that expres-
sion of DNT-MORN did not reestablish root hair formation (Figure
5D), despite full catalytic activity in vitro (Figure 2), leading us to
conclude that PIP5K3 functionality in root hair development
requires additional factors other than catalytic activity alone.
Because PtdIns(4,5)P2 has previously been described to form
a lipid microdomain in the plasma membrane of root hair tips
(Braun et al., 1999) and pollen tubes (Kost et al., 1999) and to be
generally plasma membrane associated in plant cells (van
Leeuwen et al., 2007), the plasma membrane was also an
expected localization for PIP5K3. The subcellular distribution of
overexpressed EYFP-tagged PIP5K3 suggested that the en-
zyme localizes to the extreme periphery of the apical tip region of
root hairs (Figure 8) and, with heterologous expression, also of
pollen tubes (see Supplemental Figure 4 online). In addition to a
likely plasma membrane association, EYFP-PIP5K3 also deco-
rated cytosolic particles (Figure 8D) in a pattern resembling the
distribution of exocytotic vesicles in pollen tubes (Lancelle and
Hepler, 1992; Parton et al., 2001). The fluorescence distribution
of EYFP-PIP5K3 was imaged in wild-type plants expressing the
fusion protein under the control of the EXP7 promoter fragment
also used for overexpression studies. No clear results were
obtained using a weaker intrinsic PIP5K3 promoter fragment.
Therefore, it is not clear whether the natural subcellular distribu-
tion of PIP5K3 in the root hair tip may be restricted to the plasma
membrane, to vesicles, or to both. Another possible localization
is cortical actin cytoskeletal structures. It is possible that PIP5K3
localization can change dynamically between different mem-
branes or nonmembrane locations, as has been reported previ-
ously for PIP5K1 (Im et al., 2007). The determinants of subcellular
localization of PIP5K3 thus remain a subject for future studies.
As results so far indicated that PIP5K3 was an active PI4P
5-kinase possibly localized to the plasma membrane or to
vesicles in the apical region of growing root hair cells, we tested
whether expression of PIP5K3 in pollen tubes would increase
plasma membrane levels of PtdIns(4,5)P2 (Figure 9). When
PIP5K3 and the inactive K442A were each coexpressed with
the PtdIns(4,5)P2 reporter HsPLCd1-PH, expression of the active
enzyme resulted in greatly increased plasma membrane asso-
ciation of reporter fluorescence (Figure 9C), indicating increased
levels of PtdIns(4,5)P2, consistent with our expectation based on
the localization data (Figure 8) and previous data on the topic
Figure 8. Subcellular Localization of PIP5K3 in Root Hairs.
EYFP-tagged PIP5K3 was expressed in wild-type plants under the
control of the EXP7 promoter, and the fluorescence distribution was
monitored in 8-d-old plants by confocal laser scanning microscopy.
Bright-field and fluorescence channels were imaged synchronously.
Images shown are representative for a growing root hair with vivid
cytoplasmic streaming expressing EYFP-PIP5K3 under the EXP7 pro-
moter. Bars ¼ 20 mm.
(A) Bright-field image.
(B) EYFP-PIP5K3 fluorescence.
(C) Merge of images (A) and (B).
(D) Magnified view of a 1.0-mm confocal section through the root hair
apex. The arrowhead indicates a cone-shaped distribution of EYFP-
PIP5K3–decorated cytosolic particles close to the apex of the root hair
cell.
(E) Confocal section (1.6 mm) through the apical region of a root hair, as
indicated by the dashed line in (C). Left panel, cell wall autofluorescence;
middle panel, EYFP-PIP5K3 fluorescence; right panel, merged image.
134 The Plant Cell
(Braun et al., 1999; Kost et al., 1999; Perera et al., 1999; Heilmann
et al., 2001; Dowd et al., 2006; van Leeuwen et al., 2007). By
contrast, plasma membrane fluorescence of the PtdIns(4,5)P2
reporter was much reduced when coexpressed with the inactive
K442A (Figure 9B). A low HsPLCd1-PH background signal
in pollen tubes expressing K442A can be attributed to the
presence of PtdIns(4,5)P2 formed by pollen tube endogenous
PI4P 5-kinases. While corresponding experiments with root hairs
were not successful because of high root hair autofluorescence,
the results from the heterologous pollen tube system (Figure 9)
correspond well with the in vitro characterization of PIP5K3 and
K442A (Figure 2) and with the effects of the two proteins in the
mutant complementation tests (Figure 5). Overall, the results
presented are consistent with the hypothesis that PIP5K3 pro-
duces PtdIns(4,5)P2 in the tips of growing root hair cells that is
required for growth.
A number of reported functions of PtdIns(4,5)P2 can be
envisioned to take part in the control of directional tip growth in
the apex of growing root hairs, as has been outlined in the
Introduction. The loss of cellular polarity observed in Arabidopsis
root hairs overexpressing PIP5K3 (Figure 6) is similar to pheno-
types observed with increased PtdIns(4,5)P2 levels in pollen
tubes due to inactivation of PLC (Dowd et al., 2006; Helling et al.,
2006), and pollen tubes strongly expressing the EYFP-PIP5K3
fusions also exhibited tip swelling and a loss of polar tip growth.
The observation that expression of PIP5K3 in the pip5k3-4
background resulted in nondeformed root hairs that were slightly
longer than those of wild-type plants (Figure 5G), whereas
overexpression resulted in severe deformation (Figure 6), sug-
gests that a fine balance of PIP5K3 expression may be required
for normal root hair development.
In analogy to an animal model for the involvement of
PtdIns(4,5)P2 in synaptic exocytosis (Cremona and De Camilli,
2001), a possible explanation for reduced root hair growth in the
absence of PIP5K3 is suggested by the notion that in mammalian
cells the assembly of the protein machinery required for the
fusion of PtdIns4P-coated vesicles with the plasma membrane
depends on the formation of PtdIns(4,5)P2 (Di Paolo et al., 2004;
Gong et al., 2005; Milosevic et al., 2005; Vicogne et al., 2006).
Compromised vesicle trafficking from the endoplasmic reticulum
to the Golgi due to continuous inactivation of the monomeric
GTPase, ARF1, in Arabidopsis plants has been shown to result in
reduced growth of root hairs and pollen tubes (Song et al., 2006),
similar to that seen in this study with disruption of the PIP5K3
gene. In combination with the reported accumulation of PtdIns4P-
coated exocytotic vesicles in the tip of growing root hair cells
(Preuss et al., 2006) the results presented suggest that PIP5K3
may control root hair elongation by providing PtdIns(4,5)P2 re-
quired for vesicle-to-plasma membrane fusion in the apex of the
hair cell, either at the surface of the plasma membrane target area
or that of the fusing vesicle.
A possible alternative mechanism by which plasma membrane–
associated PIP5K3 may affect root hair elongation relates to the
control of F-actin dynamics in analogy to the situation in yeast,
where the only PI4P 5-kinase, MSS4p, mediates the attachment
of F-actin to the plasma membrane and is required for yeast
budding (Desrivieres et al., 1998). In analogy to the yeast model,
PIP5K3 may yield PtdIns(4,5)P2 controlling F-actin-modulating
Figure 9. Expression of PIP5K3 in Pollen Tubes Increases Plasma
Membrane Levels of PtdIns(4,5)P2.
(A) and (B) EYFP-PIP5K3 and the catalytically inactive EYFP-K442A
protein were each coexpressed in tobacco pollen tubes with the Red-
Star-tagged HsPLCd1-PH, which specifically binds to PtdIns(4,5)P2.
EYFP and RedStar fluorescence (left and middle panels, respectively)
were synchronously recorded by confocal laser scanning microscopy
after incubation of transformed pollen for 10 h. Right panels, merged
images. Yellow color indicates colocalization of EYFP and RedStar
signals of equal intensity. Bars ¼ 10 mm.
(A) PIP5K3.
(B) K442A.
(C) Quantification of relative fluorescence intensities in horizontal sec-
tions indicated in (A) and (B). Fluorescence intensities were normalized
against the highest value for each scan, set as 1. Transient expression
was performed under identical conditions. Images were not individually
adjusted for brightness or contrast. Data presented are from a repre-
sentative experiment. Of 36 transformed pollen tubes observed for
PIP5K3 or K442A, 18 and 19 tubes, respectively, exhibited the pattern
shown. Expression levels of the remaining tubes were either too low to
visualize any RedStar fluorescence with expression of K442A or were too
high for meaningful imaging.
PI4P 5-Kinase 3 and Root Hair Development 135
proteins, such as profilin or gelsolin, that may be associated with
F-actin strands binding to PIP5K3 via its Lin domain (Davis et al.,
2007). A lack of PIP5K3 in root hairs of the Arabidopsis mutants
described may reduce directional membrane traffic by prevent-
ing F-actin polymerization at the plasma membrane.
In summary, the data presented indicate PIP5K3 as a func-
tional component of the machinery controlling directional tip
growth of root hairs. The PIP5K3 protein catalyzes the produc-
tion of PtdIns(4,5)P2 in the apical region of root hair cells and may
control vesicle trafficking and/or cytoskeletal structures required
for root hair growth. The identification of the PI4P 5-kinase
responsible for the production of PtdIns(4,5)P2 involved in root
hair growth provides a key tool needed to determine the partic-
ular physiological processes in the root hair that are controlled by
that lipid. Future work will be directed toward elucidating the
exact roles of PtdIns(4,5)P2 in the cell biology of root hair
formation.
METHODS
Plant Growth Conditions
All experiments were performed with Arabidopsis thaliana ecotype
Columbia-0 (Col-0). Seeds were surface-sterilized for 5 min in 75%
ethanol, followed by 20 min incubation in 6% (w/v) NaOCl in 0.1% (w/v)
Triton X-100, and washed five times in sterile distilled water. Before
plating, seeds were vernalized at 48C for 2 d, followed by culture on half-
strength Murashige and Skoog (MS) medium including basal salt mixture
(Duchefa) containing 1% (w/v) sucrose and 0.7% (w/v) agar (Duchefa) at
228C under continuous light.
Primers
Sequences for all primers named below are provided in Supplemental
Table 1 online.
Genotyping of T-DNA Insertion Lines
T-DNA insertion lines (pip5k3-1, SALK_060590; pip5k3-2, SALK_001546;
pip5k3-3, SALK_000024; and pip5k3-4, SALK_026683) in the Arabidopsis
Col-0 background were obtained from the Nottingham Arabidopsis Stock
Centre. A PCR-based approach was used to identify homozygous lines
and to confirm the exact insertion position of the T-DNA. For the analysis
of the pip5k3-1 mutant and pip5k3-2 mutant, primers used were LBa1,
SALK_060590_reverse, and SALK_060590_forw. For the analysis of the
pip5k3-3 mutant, primers used were LBa1, SALK_00024_reverse, and
SALK_000024_forw. For the analysis of the pip5k3-4 mutant, primers
used were LBa1, SALK_026683_reverse, and SALK_026683_forw.
Cloning of cDNA or Genomic Constructs
PI4P 5-Kinase Coding Sequences
The cDNA sequences of Arabidopsis PI4P 5-kinase genes PIP5K1, PIP5K2,
PIP5K7, PIP5K8, and PIP5K9 were amplified from cDNA prepared from
young Arabidopsis inflorescences using the following primer combinations:
PIP5K1, PIP5K1Nco_for/PIP5K1Nco_rev; PIP5K2, PIP5K2Pci_for/PIP5K2P-
ci_rev; PIP5K7, PIP5K7Nco_for/PIP5K7Nco_rev; PIP5K8, PIP5K8Nco_for/
PIP5K8Nco_rev; and PIP5K9, PIP5K9Nco_for/PIP5K9Nco_rev. The cDNA of
PIP5K3 was isolated by RT-PCR from cDNA of 14-d-old roots grown on agar
plates using the primer combination PIP5K3Nco_for/PIP5K3Nco_rev. PCR-
fragments were subcloned into the vector pGEM-Teasy (Promega) and
sequenced. The resulting plasmids were designated as PIP5K1-pGEM-
Teasy, PIP5K2-pGEM-Teasy, PIP5K3-pGEM-Teasy, PIP5K7-pGEM-Teasy,
PIP5K8-pGEM-Teasy, and PIP5K9-pGEM-Teasy, respectively.
Mutagenesis of the PIP5K3 Coding Sequence
To obtain the cDNA clone encoding K442A, the PIP5K3 coding sequence
was altered using the QuikChange site-directed mutagenesis kit (Stra-
tagene) according to the manufacturer’s instructions with the primer
combination PIPK3 K442A QC for/PIPK3 K442A QC rev. The QuikChange
product was cloned into pGEM-Teasy (Promega), yielding PIPK3-K442A-
pGEM-Teasy. The cDNA encoding TripleA was amplified from PIPK3-
pGEM-Teasy using the primer combination PIPK3_loop_for/PIPK3-
loop4_rev and cloned into pGEM-Teasy, yielding the plasmid PIPK3-
TripleA-pGEM-Teasy. To create the DNT-MORN construct, the PIP5K3
cDNA sequence omitting the cDNA encoding the N terminus of PIP5K3 was
amplified using the primer combination AtPIP5K3Nco3_for/AtPIP5K3_
rev2, and the PCR product was cloned into the vector pGEM-Teasy,
yielding the plasmid DNT-MORN-PIP5K3-pGEM-Teasy. The cDNA en-
coding the truncated pip5k3-4-protein was amplified from PIPK3-pGEM-
Teasy using the primer combination AtPIPK3_Nco_for/PIPK3_as4.
PI4P 5-Kinase Promoter Fragments
For generation of promoter-GUS fusions, the GUSPlus gene was amplified
from the vector pCAMBIA1305.1 (accession number AF354045) using the
primer combination GUS_for/GUS_rev, and the PCR product was intro-
duced as a NotI-SacI fragment into the vector pgreen0029 (http://www.
pgreen.ac.uk/) (Hellens et al., 2000), yielding the plasmid pgreenGUSPlus.
Amplification of 1500-bp genomic sequences upstream of coding se-
quences foruse aspromoterswasachievedwithdifferent ArabidopsisBAC
clone templates as indicated, using the following primer combinations:
PromPIP5K1, PromPIP5K1_for/PromPIP5K1_rev from BAC clone F2E2;
PromPIP5K2, PromPIP5K2_for/PromPIP5K2_rev from BAC clone T32E8;
PromPIP5K3, PromPIP5K3_for/PromPIP5K3_rev from BAC clone T9J22;
PromPIP5K7, PromPIP5K7_for/PromPIP5K7_rev from BACclone T19D16;
PromPIP5K8, PromPIP5K8_for/PromPIP5K8_rev from BAC clone T7P1;
and PromPIP5K9, PromPIP5K9_for/PromPIP5K9_rev from BAC clone
F8A24. The PCR products representing PromPIP5K1, PromPIP5K3,
PromPIP5K7, PromPIP5K8, and PromPIP5K9 were moved directionally
as SalI-NotI fragments into the vector pgreenGUSPlus. The PCR pro-
duct representing PromPIP5K2 was moved as a NotI-NotI fragment into
pgreenGUSPlus. The resulting plasmids were transformed into Agro-
bacterium tumefaciens strain EHA105 and used for stable Arabidopsis
transformation.
PIP5K3 Overexpression Constructs
To create PIP5K3 overexpression constructs, an 800-bp fragment of the
characterized promoter sequence of the EXP7 gene (At1g12560) (Cho
and Cosgrove, 2002) was amplified using the primer combination
PromAtEXP7_for/PromAtEXP7_rev with genomic Arabidopsis DNA as a
template. The PCR product was moved as a SalI-NotI fragment into the
vector pUC18-Entry (Hornung et al., 2005), yielding the plasmid prom-
EXP7-puc18entry. The complete cDNA sequence of PIP5K3 was ampli-
fied with the primer combination PIP5K3Nco_for/PIP5K3_rev2 using the
plasmid PIP5K3-pGEM-Teasy as a template, and the PCR product was
cloned into pGEM-Teasy. The resulting plasmid was named PIP5K3-
pGEM-Teasy2. The PIP5K3-cDNA was moved as a NotI-NotI fragment
into the vector promEXP7-puc18entry, yielding the plasmid promEXP7-
PIP5K3puc18entry. To distinguish the overexpressed PIP5K3 clone from
the endogenous PIP5K3-allele, the cDNA sequence encoding EYFP was
amplified from the plasmid pEYFP (Clontech) using the primer combination
136 The Plant Cell
EYFP-Nco_for/EYFP-Nco_rev2 and moved as an NcoI-NcoI fragment in
frame with the PIP5K3 cDNA into the plasmid promEXP7-PIP5K3-
puc18entry, yielding promEXP7-YFP-PIP5K3puc18entry. The RedStar
(Janke et al., 2004) coding sequence was amplified from plasmids
carrying the authentic clones obtained from Martin Fulda using the primer
combination Redstar_for/Redstar_rev, cloned into pGEM-Teasy, and
moved from there as a NotI-NotI fragment first into the vector prom-
EXP7-puc18entry, yielding promEXP7-Redstar-puc18entry. For overex-
pression constructs for K442A and DNT-MORN, the respective clones
were moved as NotI-NotI fragments from the corresponding pGEM-Teasy
plasmids to promEXP7-puc18entry, yielding the plasmids promEXP7-
K442A-puc18entry and promEXP7-DNT-MORN-puc18entry, respectively.
Finally, the RedStar sequence without a stop codon was amplified using
the primer combination RedStar _for/RedStar_rev2 and moved directly as
an NcoI-NcoI fragment into promEXP7-K442A-puc18entry or promEXP7-
DNT-MORN-puc18entry, respectively. All entry clones were moved into the
vector pCambia 3300 using Gateway technology (Invitrogen) according to
the manufacturer’s instructions and transformed into the A. tumefaciens
strain EHA105.
Mutant Complementation Constructs
To create constructs for mutant complementation tests, the 1500-bp
promoter fragment promPIP5K3 was moved as a SalI-NotI fragment into
the vector pUC18-Entry (Hornung et al., 2005), yielding the plasmid
promPIP5K3puc18entry. The full-length PIP5K3 cDNA sequence was
moved from PIP5K3-pGEM-Teasy2 as a NotI-NotI fragment into the
vector promPIP5K3puc18entry, yielding the plasmid promPIP5K3-
PIP5K3puc18entry. To distinguish the introduced PIP5K3 clone from
the endogenous PIP5K3 allele, the cDNA sequence encoding EYFP
was moved as an NcoI-NcoI fragment in frame with the PIP5K3 cDNA
into the plasmid promPIP5K3-PIP5K3puc18entry, yielding the plas-
mid promPIP5K3-YFP-PIP5K3-puc18entry. The DNT-MORN, K442A, or
TripleA cDNA clones were moved as NotI-NotI fragments from their
respective pGEM-Teasy plasmids into theplasmidpromPIP5K3puc18entry,
yielding the plasmids promPIP5K3-DNT-MORN-puc18entry, promPIP5K3-
K442A-puc18entry, or promPIP5K3-TripleA-puc18entry, respectively, fol-
lowed by introduction of the EYFP sequence into the NcoI site of
these plasmids to produce promPIP5K3-YFP-DNT-MORN-puc18entry,
promPIP5K3-YFP-K442A-puc18entry, and promPIP5K3-YFP-TripleA-
puc18entry. All entry clones were moved into the vector pCambia 3300
using Gateway technology (Invitrogen) according to manufacturer’s instruc-
tions and transformed into the A. tumefaciens strain EHA105.
Bacterial Expression Constructs
Constructs for heterologous expression in Escherichia coli were created
by moving the cDNA sequences encoding PIP5K1, 3, 7, 8, and 9 as NcoI-NcoI
fragments from the plasmids PIP5K1-pGEM-Teasy, PIP5K3-pGEM-Teasy,
PIP5K7-pGEM-Teasy, PIP5K8-pGEM-Teasy, and PIP5K9-pGEM-Teasy,
respectively, into the bacterial expression vector pETM-41 (EMBL Protein
Expression and Purification Facility). The cDNA sequence of PIP5K2 was
released from the plasmid PIP5K2-pGEM-Teasy as a PciI-PciI fragment
and moved into the NcoI site of pETM-41. The DNT-MORN cDNA was
moved as an NcoI-NotI fragment from the vector DNT-MORN-PIP5K3-
pGEM-Teasy into the plasmid pETM-41. The cDNA encoding the
N-terminal MORN domain (PIP5K349-226) was amplified from PIP5K3-
pGEM-Teasy using the primer combination 59-GATCCATGGAGAAGG-
TGCTAAAGAACGGC-39/59-GATCCATGGCTCTTCCTCCCCCACCC-39.
The PCR product was cloned into pGEM-Teasy and moved from that
plasmid into pETM-41 as an NcoI-NcoI fragment. The coding sequences
for K442A, TripleA, and pip5k3-4 were moved as NcoI-NotI fragments
from the respective pGEM-Teasy plasmids into pETM-41. All clones were
introduced into the pETM-41 vector in frame with the cDNA encoding the
N-terminal MBP and polyhistidine (His) tags of pETM-41.
Fusion Constructs for Particle Bombardment
The cDNA clones encoding EYFP-PIP5K3, EYFP-K442A, and EYFP-
DNT-MORN were moved as NotI-NotI fragments into the vector
pENTR2b (Invitrogen). The coding sequence for the human PLCd1-PH
domain (Varnai and Balla, 1998) was amplified from plasmid DNA pro-
vided by Tamas Balla (National Institute for Child Health and Human
Development, Rockville, MD) and modified to encode a seven–amino
acid linker (Gly-Gly-Ala-Gly-Ala-Ala-Gly) between the PH domain and the
RedStar tag, as previously described (Dowd et al., 2006), using the primer
combination 59-GATCGCGGCCGCCGGTGGAGCTGGAGCTGCAGGA-
ATGAGGATCTACAGGCGC-39/59-GATCGATATCTTAGATCTTGTGCAG-
CCCCAGCA-39. The amplicon was moved into pENTR2b as a NotI-
EcoRV fragment. The pENTR2b constructs were transferred by Gateway
technology (Invitrogen) from pENTR2b to the plasmid pLatGW, an ex-
pression vector containing the tomato Lat52 promoter (Twell et al., 1990),
an attR Gateway cassette, and a pA35S terminator. The pLatGW-vector
was a gift from Wolfgang Droge-Laser (University of Gottingen, Germany).
Detection of Specific Transcripts and Analysis of PIP5K3 Expression
Levels by RT-PCR
Expression levels of PIP5K3 in wild-type plants and in pip5k3-1, pip5k3-2,
pip5k3-3, and pip5k3-4 mutants were analyzed by RT-PCR. Total RNA
was extracted from roots, stems, leaves, and flowers using Plant RNA
purification reagent (Invitrogen). For removal of contaminating genomic
DNA, RNA samples were incubated for 30 min at 378C with RNase-free
DNaseI, and the RNA was subsequently precipitated. Five micrograms of
total RNA were reverse transcribed with RevertAid H Minus M-MuLV
reverse transcriptase (Fermentas) in the presence of oligo(dT) primers.
For determination of relative transcript levels, equal amounts of first-
strand cDNA were used as templates for PCR amplification using the
primer combination PIP5K3Nco_for/PIP5K3Nco_rev. The Arabidopsis
actin8 (ACT8) gene was amplified using the primer combination
AtACT8_F/AtACT8_R and served as an internal positive control (Bustin,
2000). Truncated transcripts as shown in Figure 5H were detected using
the primer combination YFP_s/PIPK3Nco_rev. Transcript levels shown in
Figure 7B were detected using the following primer combinations: Red-
Star, RedStar_for/RedStar_rev; K442A and DNT-MORN, RedStar-s/
PIP5K3Nco_rev.
Determination of Specific Transcript Levels by Quantitative
Real-Time RT-PCR
The levels of specific transcripts in roots were determined by real-time
RT-PCR analysis of cDNA reverse transcribed from 1 mg of total RNA
using 100 units of Reverse Transcriptase H� (MBI Fermentas) according
to the manufacturer’s instructions. The resulting cDNA was diluted 1:10,
and 1 mL was used as a PCR template in a 25-mL reaction containing 2.5
mL 103 PCR buffer (Bioline), 2 mM MgCl2, 100 mM deoxynucleotide
triphosphate, 2.5 mL QuantiTect primer mix (Qiagen), 0.1-fold Sybr green,
10 nM fluorescein, and 0.25 units of BioTaq (Bioline). Samples were
denatured for 3 min at 958C, followed by 40 cycles of 20-s denaturation at
958C, 20 s of annealing at 558C, and 40 s of elongation at 728C.
Fluorescence was monitored during each annealing and denaturation
phase. The program was concluded by 4 min of elongation at 728C and
1 min of denaturation at 958C. Renaturing of amplified DNA was followed
by the assessment of melting parameters by increasing the temperature
in 0.58C increments while monitoring fluorescence. In addition to the
individual QuantiTect primers used, a DNA fragment of the PP2A subunit
PI4P 5-Kinase 3 and Root Hair Development 137
of PDF2 (At1g13320) was used as an internal reference. Transcript levels
were calculated according to Livak and Schmittgen (2001).
Heterologous Expression in E. coli
Recombinant enzymes were expressed in E. coli strain BL21-AI (Invitro-
gen) at 258C for 18 h after induction with 1 mM isopropylthio-b-galacto-
side and 0.2% (w/v) L-arabinose. The PIP5K3-MORN domain was
expressed in E. coli Rosetta II cells (Novagen) at 258C for 4 h after
induction with 1 mM isopropylthio-b-galactoside. Cell lysates were
obtained by sonification in a lysis buffer containing 50 mM Tris-HCl,
300 mM NaCl, 1 mM EDTA, and 10% (v/v) glycerol, pH 8.0.
Lipid Kinase Assays
Lipid kinase activity was assayed by monitoring the incorporation of
radiolabel from [g-32P]ATP into defined lipid substrates according to Cho
and Boss (1995) using total extracts of BL-21-AI expression cultures.
Recombinant expression levels were adjusted between individual cul-
tures according to immunodetection of expressed MBP-tagged proteins
(data not shown), allowing for the comparison of enzyme activities
obtained with different cultures. Radiolabeled lipid reaction products
were separated by thin layer chromatography and visualized by autora-
diography using Kodak X-Omat autoradiography film (Eastman Kodak).
Reaction products were identified according to comigration with authen-
tic standards (Avanti Polar Lipids). Phosphatidylinositol-bisphosphate
bands were scraped according to autoradiography, and incorporated
radiolabel was quantified using liquid scintillation counting (Analyzer
Tricarb 1900 TR; Canberra Packard). Enzyme activites presented in
Figure 2, Table 1, and Supplemental Figure 2 online represent the mean of
at least three independent experiments, assayed in duplicates.
Arabidopsis Transformation
Recombinant constructs were introduced into Arabidopsis plants through
A. tumefaciens–mediated transformation using the floral dip method
(Clough and Bent, 1998). Independent transformants were subjected to
selective conditions on MS medium containing either 50 mg mL�1
kanamycin (for promoter-GUS plants) or 10 mg mL�1 glufosinate-ammo-
nium (for complementation constructs in T-DNA insertion lines), 1% (w/v)
sucrose, and 0.7% (w/v) agar. Resistant seedlings were transferred to soil
after 2 to 3 weeks; homozygous T2 plants were used for analysis.
Histochemical Staining for GUS Activity
Histochemical staining of plant tissue for GUS activity was performed as
previously described (Jefferson et al., 1987). In brief, tissue samples were
vacuum-infiltrated for 5 min in a GUS substrate solution of 100 mM
sodium phosphate, pH 7.0, 2 mM 5-bromo-4-chloro-3-indolyl-glucu-
ronide, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide,
10 mM EDTA, and 0.1% (v/v) Triton X-100 and then incubated at 378C for
3 h. Subsequently, the samples were transferred to 70% ethanol to
remove chlorophyll pigmentation. GUS-positive samples were examined
with a bright-field microscope (Olympus BX51) or a stereomicroscope
(Olympus SZX12) at low magnification (34 to 310), and digital images
were recorded. For cross sections, stained roots were embedded in
polyethylene glycol 1500 according to Hause et al. (1996). Cross sections
of 10 mm thickness were cut with a microtome (HM 355; Microm
International), transferred to poly-L-Lys–coated slides, and examined by
bright-field microscopy using an AxioImager microscope (Zeiss). Micro-
graphs taken with AxioCam MRc5 (Zeiss) were processed through
Photoshop 8.0.1 (Adobe Systems). All GUS-stained samples shown
represent typical results of at least three independent transgenic lines for
each construct.
Quantification of Root Hair Length
Root hair length from 8-d-old plants grown on agar plates was deter-
mined on low-magnification (310) digital images captured using a CCD
camera (ColorViewII) and image analysis freeware (ImageJ; http://rsb.
info.nih.gov/ij/). To ensure comparable results, the area 3 to 5 mm behind
the root tip was analyzed. Plants grown on agar plates were carefully
removed in ;100 mL of half-strength MS medium (Duchefa) on micro-
scope slides for analysis. Quantification data are the means of 200 to 350
values representing 10 root hairs each of 20 to 35 individual plants
measured for each data point.
Pollen Tube Growth and Transient Gene Expression
Mature pollen was collected from four to six tobacco (Nicotiana tabacum)
flowers of 8-week-old plants. Pollen was resuspended in growth medium
(Read et al., 1993), filtered onto cellulose acetate filters, and transferred to
Whatman paper moistened with growth medium. Within 5 to 10 min of
harvesting, pollen was transformed by bombardment with plasmid-
coated 1-mm gold particles with a helium-driven particle accelerator
(PDS-1000/He; Bio-Rad) using 1350-psi rupture discs and a vacuum of
28 inches of mercury. Gold particles (1.25 mg) were coated with 3 to 7 mg
of plasmid DNA. After bombardment, pollen was resuspended in growth
medium and grown for 7 to 10 h in small droplets of media directly on
microscope slides.
Microscopy and Imaging
Images were recorded using a Zeiss LSM 510 confocal microscope.
EYFP was excited at 514 nm and imaged using an HFT 405/514/633-nm
major beam splitter (MBS) and a 530 to 600-nm band-pass filter; RedStar
was excited at 561 nm and imaged using an HFT 405/488/561 nm MBS
and a 583 to 604-nm band-pass filter; EYFP and FM4-64 were synchro-
nously excited at 488 nm and 561 nm, respectively, and imaged using an
HFT 405/488/561-nm MBS and a 518 to 550-nm band-pass filter and a
657 to 754-nm band-pass filter, respectively. Root hair autofluorescence
was imaged using an HFT 405/514/633-nm MBS and a 470 to 500-nm
band-pass filter to record autofluorescence without fluorescence cross-
leakage from the EYFP channel. The autofluorescence signal was then
subtracted from the EYFP signal. Fluorescence and transmitted light
images were contrast-enhanced by adjusting brightness and g-settings
using image-processing software (Photoshop; Adobe Systems), except
where stated differently. Fluorescence intensities were determined using
AnalySIS software (Olympus). FM4-64 was added to pollen tubes at a
final concentration of 10 mM as described (Parton et al., 2001) and
visualized after 5 to 15 min of incubation before dye internalization.
Accession Numbers
Arabidopsis Genome Initiative locus identifiers of genes used in
this study are as follows: PIP5K1, At1g21980; PIP5K2, At1g77740;
PI5K3, At2g26420; PIP5K7, At1g10900; PIP5K8, At1g60890; PIP5K9,
At3g09920; ACT8, At1g49240; PDF2 (PP2A), At1g13320.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Organ-Specific Expression Patterns of PI4P
5-Kinase Isoforms 2 and 9 in Arabidopsis.
Supplemental Figure 2. Catalytic Activity of Arabidopsis PI4P
5-Kinase Isoforms Expressed in Roots.
Supplemental Figure 3. Identification of Homozygous T-DNA Inser-
tion Mutants in the PIP5K3 Gene Locus At2g26420.
138 The Plant Cell
Supplemental Figure 4. Subcellular Localization of PIP5K3 and
K442A Heterologously Expressed in Tobacco Pollen Tubes.
Supplemental Table 1. Primers Used in This Study.
Supplemental Text and References.
ACKNOWLEDGMENTS
We thank the following individuals: Tamas Balla (National Institute for Child
Health and Human Development, Rockville, MD) for the HsPLCd1-PH
domain construct, Andreas Hiltbrunner (Eidgenossische Technische
Hochschule, Zurich, Switzerland) for the pCambia vector, Wolfgang
Droge-Laser and Martin Fulda for cDNA constructs, Susanne Mesters
for expert plant culture, Andreas Wodarz and Michael Krahn for access
to the confocal microscope and technical support, respectively, Linh Vu
Hai for technical assistance (all University of Gottingen, Germany). We
also thank Imara Perera, Wendy Boss, and Yang Ju Im (all North
Carolina State University, Raleigh, NC) and Ivo Feussner (University of
Gottingen, Germany) for helpful discussion and critical reading of
this manuscript. We gratefully acknowledge financial support by the
ERASMUS/SOCRATES program (to A.H. and M.S.) and an Emmy
Noether grant from the German Research Foundation (to I.H.).
Received May 15, 2007; revised December 13, 2007; accepted Decem-
ber 18, 2007; published January 4, 2008.
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PI4P 5-Kinase 3 and Root Hair Development 141
DOI 10.1105/tpc.107.052852; originally published online January 4, 2008; 2008;20;124-141Plant Cell
HeilmannIrene Stenzel, Till Ischebeck, Sabine König, Anna Holubowska, Marta Sporysz, Bettina Hause and Ingo
Arabidopsis thalianaThe Type B Phosphatidylinositol-4-Phosphate 5-Kinase 3 Is Essential for Root Hair Formation in
This information is current as of January 9, 2021
Supplemental Data /content/suppl/2007/12/20/tpc.107.052852.DC1.html
References /content/20/1/124.full.html#ref-list-1
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